Chondroinductive Peptides for Cartilage Regeneration

Abstract

Inducing and maintaining a hyaline cartilage phenotype are the greatest challenge for cartilage regeneration. Synthetic chondroinductive biomaterials might be the answer to the unmet clinical need for a safe, stable, and cost-effective material capable of inducing true hyaline cartilage formation. The past decade witnessed an emergence of peptides to achieve chondrogenesis, as peptides have the advantages of versatility, high target specificity, minimized toxicity and immunogenicity, and ease of synthesis. In this study, we review peptides as the basis for creating promising synthetic chondroinductive biomaterials for in situ scaffold-based cartilage regeneration. We provide a thorough review of peptides evaluated for cartilage regeneration while distinguishing between peptides reported to induce chondrogenesis independently, and peptides reported to act in synergy with other growth factors to induce cartilage regeneration. In addition, we highlight that most peptide studies have been in vitro, and appropriate controls are not always present. A few rigorously performed in vitro studies have proceeded to in vivo studies, but the peptides in those in vivo studies were mainly introduced through systemic, subcutaneous, or intra-articular injections, with a paucity of studies employing in situ defects with appropriate controls. Clinical translation of peptides will require the evaluation of these peptides in well-controlled in vivo cartilage defect studies. In the decade ahead, we may be poised to leverage peptides to design devices that are safe, reproducible, cost-efficient, and scalable biomaterials, which are themselves chondroinductive to achieve true hyaline cartilage regeneration without the need for growth factors and other small molecules.

Impact statement

The regeneration of articular cartilage into its original structural, functional, and organizational hyaline phenotype remains a significant problem in the tissue engineering and orthopedic community. While cell-based solutions have shown promising outcomes, there are realistic translational challenges inherent to cell therapies. Alternatively, biomaterials have been widely studied and used as scaffolds to support and facilitate cartilage regeneration; however, the key technical challenge is to independently induce cartilage regeneration. The search for chondroinductive compounds and materials is an emerging area of research with peptides at its heart, which presents a timely opportunity to review and highlight peptides with cartilage regenerative activity and to fill gaps from previous reviews. The content of this review will serve as a valuable guide for researchers pursuing the discovery of new chondroinductive peptides or looking into incorporating the most promising existing peptides in their work.

Introduction

Articular cartilage regeneration manifests as a tough challenge for researchers and clinicians globally. The loss of cartilaginous tissues affects all age groups and is mainly due to arthritis and traumatic injuries. According to the 4th edition of the Burden of Musculoskeletal Diseases in the United States (BMUS), published in 2018, ∼78 million Americans will develop arthritis by 2040, and the annual direct medical costs attributed to arthritis are roughly $81 billion in the United States.¹

Articular cartilage cannot self-regenerate mainly due to the lack of vascularization and the low density of chondrocytes. Nonsurgical treatments include intra-articular injections, with standard examples such as corticosteroids or hyaluronic acid (HA; FDA approved). However, intra-articular injections are an advanced pharmacological intervention to manage the pain for patients with persistent osteoarthritis (OA) symptoms and are typically used as a last resort before or in lieu of surgical intervention. A recent review by Jones et al.² highlighted the currently available intra-articular injections for knee OA and emphasized the need to have significant clinical data to support the effectiveness of these treatments compared to placebo.

Currently available surgical approaches include microfracture (MF), which was introduced in the early 1980s³ and is still one of the first choices for treating cartilage injury due to its feasibility and lower cost compared to alternative surgical approaches. However, MF is mainly employed for small defects, and the repair tissue is usually fibrocartilage. Other options include autologous osteochondral grafts (mosaicplasty) and osteochondral allografting (OCA). Mosaicplasty is becoming less popular due to donor site morbidity and variability of outcomes⁴; as for OCA, the main drawbacks are cost and graft availability.⁵

Regenerative surgical techniques emerged ∼30 years ago^6,7 with the primary technique being autologous chondrocyte implantation (ACI). ACI has been modified for second, third, and fourth generation.^7,8 The third generation refers to the matrix-assisted chondrocyte implantation (MACI), involves the use of scaffolds, and in December 2016,⁹ the FDA approved Vericel's MACI^® for full-thickness cartilage defects.

Clinical outcomes of regenerative surgical techniques are affected by several variables such as, lesion size, site of defect, sex, and age; however, most reviews addressing MF, ACI, and MACI conclude that ACI or MACI are recommended when lesions are >3 cm.^10,11 Fourth-generation ACI overcomes the limitation of two surgical procedures in previous generations, and involves chondrocyte and bone marrow stem cell (BMSC) harvest and implantation in one intervention.⁷ Single treatment autologous chondrocyte implantation (STACI) and INSTRUCT¹² are currently available fourth-generation ACI techniques; however, as of May 2021, they were not FDA approved.

However, despite the pain relief and enhancement of knee function, the outcome of regenerative surgical techniques in most cases is an inferior fibrous tissue that lacks the structural organization, matrix composition, and mechanical properties of hyaline cartilage. Therefore, the question that remains is, how can we induce true hyaline cartilage regeneration? Several approaches to answer this question have been extensively studied by the tissue engineering community, which is reflected by the number of reviews that have been published in the past couple of years to address cell therapy,^4,13–22 scaffolds,^4,23 biomaterials,^16,24,25 hydrogels,^11,26–28 three-dimensional (3D) printing,²¹ and gene therapy,¹⁷ and so on.

However, there remains an unmet clinical need for the development of small synthetic molecules that have the potential to induce chondrogenesis (i.e., chondroinductive) and promote cartilage regeneration without the fear of immunogenicity of naturally derived components, the high cost of surgical and cell therapy procedures, and the potential variability of extracellular matrix (ECM) products. Such device-only, synthetic chondroinductive materials would provide a safe, cost-effective, and translational approach toward successful true hyaline cartilage regeneration.

Two main promising categories of synthetic chondroinductive materials are currently recognized, small molecules and peptides, of which peptides will be the focus of this review. Chondroinductive pathways of stem cells are initiated through protein-protein interactions (i.e., between cell surface receptors, growth factors, and ECM proteins). The small binding pocket of the cell membrane protein or the binding ligand of the exogeneous protein may be identified and generated as a peptide to trigger a given pathway, even without the ligand protein. The particular binding pocket may additionally be triggered with small molecules, only if the binding site has a defined “hot spot” to target.²⁹ However, protein-protein interactions can be wide and have more than one “hot spot” to target. Therefore, many protein-protein interactions remain as “undruggable” by the small molecules.³⁰ Peptides, by covering the wide binding pocket and interacting with the “hot spots,” may be more specific and more effective.³¹

Small molecules have been widely investigated for their ability to induce stem cell differentiation and chondrocyte proliferation, and to maintain chondrocyte proliferation. A recent review by Li et al.³² provided an extensive list of natural and synthetic small molecules that have been evaluated for their applications in cartilage tissue engineering and regeneration. Among the reported synthetic small molecules, KA-34³³ (kartogenin analog) and lorecivivint^34–38 (SM04690) may perhaps be the most promising so far. Lorecivivint is already in phase 3 clinical trials as a disease-modifying osteoarthritic drug (DMOAD),³⁹ whereas KA34 has a phase 1 trial completed, but no results or plans for phase 2 are yet published.

On the other hand, several peptides have been studied for their chondroinductive potential, and a few reviews^40–42 have addressed the use of these peptides. A previous review from our group provided a concise review of chondrogenic peptides related to cell adhesion sequences.⁴² Liu et al.⁴⁰ focused on the different applications of peptides in cartilage regeneration and distinguished peptides based on their function (e.g., transforming growth factor [TGF]-β mimics, affinity, cell-penetrating, self-assembly, and degradable peptides).⁴⁰ Gonzalez-Fernandez et al.⁴¹ focused on materials used for musculoskeletal regeneration and listed peptides that can be employed for chondrogenesis, osteogenesis, and myogenesis.⁴¹

However, the previous reviews did not provide a clear distinction between peptides that are chondrogenic and those that facilitate or synergize with other factors to induce chondrogenesis. In addition, none of the previous reviews highlighted the in vitro controls and animal models used to evaluate the chondroinductive potential of peptides.

This review provides a comprehensive overview of peptides involved in cartilage regeneration, with an emphasis placed on the in vitro and in vivo controls, animal models, and defect types used to evaluate the peptides. Figure 1 presents an overview of the different sources of peptides reported to be used for cartilage regeneration. In addition, we divide peptides into two categories, the first includes peptides reported to be chondroinductive without the addition of growth factors in vitro or in vivo. The second category includes peptides that were reported to be used in cartilage regeneration, but where chondrogenesis was only observed in the presence of growth factors. Tables 1 and 2 provide a summary of in vivo and in vitro studies, respectively, for peptides reported to exhibit chondroinductive activity. Tables 3 and 4 provide a summary of in vivo and in vitro studies, respectively, for peptides reported to be used in cartilage regeneration.

FIG. 1.

Different categories of peptides for cartilage regeneration with a nonexhaustive list of example peptides in each category. (A) Peptides derived from or inspired by growth factors that play a role in chondrogenesis or peptides, which are designed to bind to growth factor receptors to activate chondroinductive-related pathways. (B) Peptides designed to bind to growth factors or recruit MSCs, usually used in combination with scaffolds to induce chondrogenesis through endogenous cells and signals. (C) Peptides derived from ECM components such as collagen I, II, Link N; or peptides that bind to glycosaminoglycans. Beyond these three general categories, self-assembling peptides represent a separate approach as a scaffolding material. Citations to individual peptides may be found in Tables 1–4. BMPs, bone morphogenetic proteins; ECM, extracellular matrix; IGF, insulin-like growth factors; MSC, mesenchymal stem cell; TGF, transforming growth factors. Color images are available online.

Table 1.

Summary of In Vivo Studies Performed with Chondroinductive Peptides

Peptide	Parent/target molecule	References	Sham	Negative/material control	Positive control	Growth factors added	Animal model	Defect type and size	Cells included	Injection method	Evidence of chondroinduction
CK2.1	Inhibits the binding of CK2 to BMPRIa	Akkiraju et al. (2017a)⁴³	None	PBS injection	BMP-2	None	C57BL/6J mice	N/A	None	Systemic	Enhanced articular cartilage formation based on Saf-O/fast green staining and col-II and IX immunostaining (N, P).
CK2.1	Inhibits the binding of CK2 to BMPRIa	Akkiraju et al. (2017b)⁴⁴	Yes	PBS injection	None	None	C57BL/6J mice	DMM	None	Intra-articular	Increased immunostaining of col-II and IX along with a low immunostaining of col-X and osteocalcin (M, N).
HSNGLPL	TGF-β1 affinity	Shah et al.⁴⁵	None	None	(1) rhTGF-β1 (2) TGFBPA+rhTGF-β1	None	Rabbits	Two 2 mm diameter full-thickness chondral defects followed by MFX were created in the femoral trochlear groove	None	Self-assembly supramolecular nanofibers (TGFBPA) fitted into defects	Significant enhancement of the regenerative potential of MFX-treated chondral defects based on gross morphology, col-II immunostaining, and GAG staining by Saf-O (M, P).
HSNGLPL	TGF-β1 affinity	Chen et al.⁴⁶	Yes	Chitosan scaffold	None	None	Rabbits	4 mm Diameter osteochondral defects in the middle of the femoral trochlear groove	None	Sponges press-fit into defects	Cartilage surface of peptide-treated groups was smooth and continuous with higher GAG and col-II staining (Sh, M).
HAVDI-	N-cadherin	Bian et al.⁴⁷	None	MeHA hydrogel group and scrambled group	None	TGF-β3-loaded microspheres	Nude mice	N/A	hMSCs	Subcutaneous injection	Peptide-containing implants exhibited a higher content of GAGs and collagen, and more intense staining of col-II and CS in the presence of hMSCs and TGF-β3-loaded microspheres (M, S).
		Feng et al.⁴⁸	Yes	Aggrecanase 1 degradable hydrogel	None	None	Rabbits	4 mm Diameter osteochondral defects in the patellar groove	Rabbit BMSCs	Hydrogels implanted into defects	HAV-conjugated hydrogels exhibited more intense staining of GAGs and col-II and no col-I staining (M, S).
		Teng et al.⁴⁹	None	MeHA hydrogel	None	None	Nude mice	N/A	hBMSCs	Subcutaneous injection	MeHA+HAV+RGD hydrogels exhibited enhanced GAG and collagen content (M). MeHA+HAV+RGD hydrogels with encapsulated kartogenin exhibited a high and significant increase in GAG and collagen content compared to MeHA+HAV+RGD.
CM-10 (LIANAK)	TGF-β1	Zhang et al.⁵⁰	None	Microspheres	None	None	Mice	N/A	Rabbit BMSCs	Subcutaneous injection	Ectopic cartilage formation was observed as demonstrated by intense Saf-O staining of GAGs and col-II immunostaining, with no apparent mineralization (M).
CM-10 (LIANAK)	TGF-β1	Park et al.⁵¹	None	Hydrogel alone	None	None	Mice	N/A	±hPLSCs	Subcutaneous injection	CM-10 resulted in significant chondrogenic differentiation with increased staining of col-II and GAGs. Increased gene expression of SOX9, ACAN, and col-II (M).
B2A	BMP-2 receptors	Lin et al.⁵²	None	Saline injection	Normal	None	Rats	Chemically induced OA by MIA	None	Synovial space injection	Significant repair of articular cartilage in B2A-treated group based on H&E and Saf-O staining (N).

M: compared to material control; N: compared to negative control; P: compared to positive control; S: compared to scrambled group; Sh: compared to sham.

ACAN, aggrecan; BMP, bone morphogenetic protein; BMPRIa, bone morphogenetic protein receptor type Ia; BMSCs, bone marrow stem cells; CK2, casein kinase 2; CM, cytomodulin; CS, chondroitin sulfate; DMM, destabilization of the medial meniscus; GAGs, glycosaminoglycans; HAV, His-Ala-Val; H&E, hematoxylin and eosin; hBMSCs, human bone marrow stem cells; hMSCs, human mesenchymal stem cells; hPLSCs, human periodontal ligament stem cells; MeHA, methacrylated hyaluronic acid; MFX, microfracture; MIA, monoiodoacetate; OA, osteoarthritis; Saf-O, Safranin O; PBS, phosphate buffered saline; TGF, transforming growth factor; TGFBPA, TGF-binding PA.

Table 2.

Summary of In Vitro Studies Performed with Chondroinductive Peptides

Peptide	Parent molecule	References	Format	Cells	Negative/material control	Positive control	Growth factors added	Evidence of chondroinduction
CK2.1	Inhibits the binding of CK2 to BMPRIa	Akkiraju et al. (2017a)⁴³	Soluble peptide	C3H10T1/2 stem cells	Peptide-free medium	BMP-2	None	CK2.1 stimulated an increase in proteoglycan and col-II synthesis, along with lower gene expression of col-X and osteocalcin (N, P).
HSNGLPL	TGF-β1	Shah et al.⁴⁵	PA gels	Human MSCs	Filler PA	Filler PA+TGF-β1	TGF-β1	HSNGLPL treatment resulted in a higher ACAN gene expression level after 4 weeks of culture, but not at previous time points (M). After 3 weeks, there was no difference in GAG production between peptide and control groups (M).
HSNGLPL	TGF-β1	Chen et al.⁴⁶	Chitosan scaffold (CHI)	Pig MSCs	Chitosan scaffold	CHI+TGF-β1	Preloaded with TGF-β1	Scaffolds with 10:3 chitosan to peptide ratio exhibited the highest gene expression levels of SOX9, col-II, and ACAN, whereas col-X was not upregulated (M, P).
HAVDI-	N-cadherin	Bian et al.⁴⁷	MeHA hydrogel+peptide	Human MSCs	MeHA hydrogel and scrambled	None	TGF-β3	HAVDING significantly increased the gene expression of col-II, ACAN, and SOX9 on days 1 and 3; however, by day 7, no significant difference was observed between groups. Following 28-day culture with hMSCs, HAVDING increased GAG and collagen content (M, S).
		Kwon et al.⁵³	MeHA hydrogel+HAV motif with ADAM10-cleavable domain	Human MSCs	MeHA hydrogel group and scrambled peptide group	None	TGF-β3	HAVDING treatment resulted in a dose-dependent increase in col-II gene expression during the first 7 days of culture, but not at 14 days. At 56 days, increased levels of sGAGs and col-II staining was observed (M, S).
		Eren Cimenci et al.⁵⁴	Amphiphilic peptide nanofiber system+HAV motif	Rat BMSCs	Amphiphilic peptide nanofiber system+nonbioactive motif and TCP	None	StemPro Chondrogenesis Differentiation Kit	Increased GAG accumulation was observed in rBMSCs cultured on HAV-nanofibers (M). Increased gene expression of col-II, ACAN, and SOX9 was observed on days 3, 7, and 14 in rBMSCs cultured on HAV-nanofibers (M).
		Teng et al.⁴⁹	Crosslinked with MeHA hydrogel	hBMSCs	MeHA hydrogel	None	TGF-β3	MeHA+HAV+RGD significantly upregulated the gene expression of ACAN, and col-II compared to MeHA and MeHA+HAV. The encapsulation of kartogenin in MeHA+HAV+RGD resulted in the highest gene expression of ACAN, col-II, and SOX9 compared to MeHA+RGD+HAV.
SPPEPS	Aggrecan and TGF-β3	Mahzoon et al.⁵⁵	Soluble or with PHA hydrogel	Rat BMSCs	Peptide-free group	TGF-β3 (proteomic analysis only)	None	SPPEPS in soluble form enhanced the expression of SOX9 and col-II after 3 days of culture (M). Proteomic analysis showed 60 proteins common between the SPPEPS and positive control groups on day 7. On day 14, 26 proteins were shared between the SPPEPS group and the positive control group.
CM-10 (LIANAK)	TGF-β1	Zhang et al.⁵⁰	Nanofibrous microspheres+CM-10	Rabbit BMSCs	Nanofibrous microspheres	None	None	Positive Saf-O staining, appearance of round differentiated cells encased in lacunae (M).
CM-10 (LIANAK)	TGF-β1	Park et al.⁵¹	Soluble or crosslinked with HA hydrogel	hPLSCs	Peptide and growth factor free	TGF-β	None	CM-10 increased the staining of col-II and GAGs (N). CM-10 increased gene expression of SOX9, ACAN, and col-II at a comparable fold increase to TGF-β group (N, P).
LINK N	Hyaluronate and aggrecan	He et al.⁵⁶	Soluble peptide	CSPCs	Peptide and growth factor free	TGF-β3	None	Increased concentrations of Link N protein resulted in increased gene expression of SOX9, col-II, and ACAN with no increase in Runx2 or col-X expression. Link N stimulated the chondrogenic differentiation of CSPCs in 3D culture, based on col-II, SOX9 immunostaining. The best performance was for the peptide+TGF-β3 group (N, P).
GFOGER (CMP)	Binds α2β1 integrin	Liu et al.⁵⁷	PEG hydrogel+peptide	Human MSCs	Peptide-free hydrogel	TGF-β3	±TGF-β3	CMP enhanced GAG, col-II, and ACAN staining in the presence of TGF-β3 (M). SOX9 gene upregulated, col-X gene downregulated (M).
		Mhanna et al.⁵⁸	PEG hydrogel with MMP-sensitive motifs+peptide	Human MSCs	Peptide-free hydrogel	None	TGF-β3	GFOGER increased GAG and DNA synthesis. Gene expression of col-II was highest in GFOGER-modified degradable gels (M).
B2A	BMP-2 receptors	Lin et al.⁵⁹	Soluble peptide	C3H10T1/2hBMSCsHuman NACs	Peptide-free culture medium	Only in the col-II experiment	None	B2A upregulated gene expression of FGFR1, FGFR2, Fgf1, Smad1, Smad4, and Twist1 (N). Matrix genes col-I and col-II were upregulated (N). B2A stimulated the production of GAGs and collagen II in hBMSCs and human chondrocytes in micromass culture (N).
KIPKASSVPTELSAISTLYL	BMP-2	Renner et al.⁶⁰	Soluble peptide	Human MSCs	Peptide-free culture medium	BMP-2; TGF-β3	None	BMP peptide increased the production of GAGs (N, P). Significant increase in total collagen. No increase in AP activity or col-I deposition (P). Upregulated gene expression of col-II and SOX9 at week 1 (N). Col-I and X gene expression increased as well (N), but at reduced values compared to BMP-2-treated cells.
RYPISRPRKR	HA-bind	Parmar et al.⁶¹	MMP7-Scl2	Human MSCs	Peptide-free hydrogel; scrambled peptide hydrogel	None	TGF-β3	HAbind and CSbind enhanced the gene expression of col-II, ACAN, and SOX9 in hMSCs (M). The highest gene upregulation of chondrogenic markers was for HAbind at 10% functionalization group. Col-I and X gene expression were significantly lower in HAbind and CSbind hydrogels (M). Total collagen, sGAG, and DNA content were highest for the HAbind (10%) groups (N, P).
YKTNFRRYYRF	CS-bind
GRVDWLQRNANFYDWFVAELG	IGF-1	Renner and Liu⁶²	Soluble peptide	Human MSCs	Peptide and growth factor-free medium	TGF-β3	Insulin and TGF-β3	In the presence of insulin and TGF-β3, the insulin peptide treatment resulted in an increased production of GAG compared to the TGF-β3-positive control.

M: compared to material control; N: compared to negative control; P: compared to positive control; S: compared to scrambled group.

3D, three dimensional; AP, alkaline phosphatase; CMP, collagen mimetic peptide; CSPCs, cartilage stem/progenitor cells; FGFR1, fibroblast growth factor receptor 1; Fgfr2, fibroblast growth factor receptor 2; HA, hyaluronic acid; MMP, matrix metalloproteinase; MSCs, mesenchymal stem cells (tissue source not specified); NAC, normal articular chondrocytes; PA, peptide amphiphile; PEG, poly(ethylene glycol); PHA, pentenoate-functionalized hyaluronic acid; rBMSCs, rat bone marrow stem cells; Scl2, recombinant streptococcal collagen-like 2; TCP, tissue culture plate.

Table 3.

Summary of In Vivo Studies for Peptides Reported to Be Used for Cartilage Regeneration

Peptide	Parent/target molecule	References	Sham	Negative/material control	Positive control	Growth factors added	Animal model	Defect type and size	Cells included	Injection method	Evidence of chondroinduction
WYRGRL	Col-II	Hesse et al.⁶³	None	StarPEG/heparin hydrogels alone	None	None	Mice	N/A	Chondrocytes	Subcutaneous injection	Not attractive to induce collagen deposition (M).
KLER	Decorin	Hesse et al.⁶³	None	StarPEG/heparin hydrogels	None	None	Mice	N/A	Chondrocytes	Subcutaneous injection	Not attractive to induce collagen deposition (M).
EPLQLKM (E7)	MSC affinity peptide	Huang et al.⁶⁴	None	MFX/DBM+Chitosan scaffold	None	None	Rabbits	Full-thickness osteochondral defects (4 mm diameter, 2.5 mm deep) were created on the trochlear groove	None	DBM (4 mm diameter, 2 mm height) was implanted into the osteochondral defects followed by injection of preprepared chitosan solution	At 24 weeks, E7-containing scaffolds showed superior cartilage repair quality and quantity based on IHC, toluidine blue staining, and did not exhibit hypertrophic cartilage remodeling (M, N).
EPLQLKM (E7)	MSC affinity peptide	Meng et al.⁶⁵	None	DBM+Chitosan scaffold/Chitosan scaffold	None	Scaffolds precultured in chondrogenic medium	Mice	N/A	BMSCs	Injection into fossa iliaca subcutaneous region	E7-containing scaffold exhibited translucent and superior cartilage-like structures, based on gross observation, H&E, toluidine blue and col-II staining (M).
KLD	Self-assembling peptide	Miller et al.⁶⁶	Yes-contralateral untreated control	None	None	None	Rabbits	Full-thickness, critically sized defect (3 mm diameter × 2 mm deep) in the central region of the femoral trochlear groove	± BMSCs	Peptide solution directly added to defects followed by Lactated Ringer's Solution to polymerize into the hydrogel	KLD hydrogel alone resulted in a significantly higher Saf-O, col-II immunostaining, and cumulative histology scores compared to KLD hydrogels containing chondrogenic factors or BMSCs (Sh).
KLD	Self-assembling peptide	Miller et al.⁶⁷	Yes	± MFX	None	None	Horses	15 mm Diameter defect in the medial trochlear ridge	None	Peptide solution directly added to defects followed by Lactated Ringer's Solution to polymerize into the hydrogel	KLD-treated defects exhibited decreased ACAN and col-II staining in the proximal side of the repair tissue (Sh). The combination of MFX+KLD presented no additional improvement compared to MFX- or KLD-only treatment, and it resulted in decreased col-II staining compared with MFX-only treatment.
(RADA16-I)/PFS (PFSSTKT)	Self-assembling peptide/bone marrow homing peptide	Lu et al.⁶⁸	MFX	(1) RAD alone(2) ACM-RAD	None	None	Rabbits	Cylindrical full-thickness defect (3 mm in diameter × 1.5 mm depth) in the center of the trochlear groove	None	Implants were placed flush with the surface of the surrounding cartilage	ACM-KLD/PFS exhibited an increased gene expression of aggrecan, SOX9, and col-II, with the highest increase being for col-II (Sh, M).

M: compared to material control; N: compared to negative control; P: compared to positive control; S: compared to scrambled group; Sh: compared to sham.

ACM, acellular cartilage matrix; DBM, demineralized bone matrix; IHC, immunohistochemistry; RAD, Ac-(RADA)4-NH2.

Table 4.

Summary of In Vitro Studies for Peptides Reported to Be Used for Cartilage Regeneration

Peptide	Parent molecule	References	Format	Cells	Negative/material control	Positive control	Growth factors added	Outcome
ANVAENA (CM-1)	TGF-β1	Renner and Liu⁶²	Soluble	Human MSCs	Peptide and growth factor-free medium	TGF-β3	None	ANVAENA treatment resulted in much lower GAG content (P). No significant increase compared to the negative control.
GPPDWHWKAMTH (R1-P1)	FGFR1	Tan et al.⁶⁹	Soluble	ATDC5 and chondrocytes	Peptide-free medium	PD173074, FGFR1 inhibitor	None	Intra-articular injection of R1-P1 was reported to significantly decreased the cartilage destruction and IL-1β-induced proteoglycan loss in an OA mouse model.
WYRGRL	Col-II	Hesse et al.⁶³	StarPEG/heparin hydrogels functionalized with CWYRGRL	Human MSCs	Peptide-free hydrogels	None	TGF-β1	StarPEG/Heparin hydrogels functionalized with CWYRGRL induced a significant decline of MSC proliferation and chondrogenic markers (SOX9, BGN, and col-II) gene expression (N).
KLER	Decorin	Salinas and Anseth⁷⁰	PEG scaffold functionalized with RGD and KLER	Human MSCs	Scrambled peptide group	None	±TGF-β1	KLER promotes chondrogenesis of MSCs when combined with RGD in PEG scaffold in chondrogenic medium.
KLER	Decorin	Hesse et al.⁶³	StarPEG/heparin hydrogels functionalized with KLER	Human MSCs	Peptide-free hydrogels	None	TGF-β1	StarPEG/Heparin hydrogels functionalized with KLER did not promote advanced col-II deposition (N).
GTPGPQGIAGQRGVV (P15)	Col-I	Zhang et al.⁷¹	Coated on culture dishes	C3H10T1/2	Uncoated dishes	None	TGF-β (not specified if 1 or 3)	P15 enhanced cell commitment to the chondrocyte lineage. P15 may serve to increase the attachment of mesenchymal cells to the matrix and then, in response to BMP-2/growth factors, leads to increased chondrogenic differentiation and an earlier start to endochondral bone formation.
CDPGYIGSR	Laminin	Kuo and Wang⁷²	CDPGYIGSR-modified scaffolds PEO and chitosan	BKCs	Peptide-free scaffolds	None	None	CDPGYIGSR-modified scaffolds supported the proliferation of BKCs. Increased content of GAGs and collagen was reported (M).
EPLQLKM (E7)	MSC affinity peptide	Meng et al.⁶⁵	E7-modified DBM particles and Chitosan scaffold	Rat BMSCs	(1) Chitosan scaffold(2) Chitosan/DBM scaffold	None	None	E7 increased the gene expression of col-II and ACAN. Increased staining of GAGs was observed (M).
RADA	N/A	Kopesky et al.⁷³	Self-assembling peptide	BMSCs	(1) Agarose hydrogel(2) TGF-β1-free medium	None	±TGF-β1	RADA hydrogels upregulated the gene expression of ACAN, SOX9, and col-II along with no upregulation of col-I in the presence of TGF-β1 (M). A higher cell number was observed with RAD16-I and KLD12 peptide hydrogels compared to agarose.
KLD	N/A	Kopesky et al.⁷³	Self-assembling peptide	BMSCs	(1) Agarose hydrogel(2) TGF-β1-free medium	None	±TGF-β1

M: compared to material control; N: compared to negative control; P: compared to positive control; S: compared to scrambled group.

BKCs, bovine knee chondrocytes; PEO, containing polyethylene oxide.

Finally, we aim from this review to highlight the currently existing peptides capable of inducing chondrogenesis and to emphasize the gaps that need to be filled to drive the field of synthetic chondroinductive biomaterial devices forward.

CK2.1 (QIKIWFQNRRKWKKMVPSDPSYEDMGGC)

In 2017, two studies were reported by Akkiraju et al.,^43,44 which addressed the regenerative potential of peptides inspired by the protein-protein interaction between the protein casein kinase 2 (CK2) and the bone morphogenetic protein (BMP) receptor type Ia (BMPRIa). CK2 is bound to the intracellular domain of BMPRIa, but is released inside the cell when BMPRIa binds to BMP-2, with the release of CK2 playing a role in downstream signaling in carrying the BMP-2 signal forward. CK2.1, CK2.2, and CK2.3 peptides were designed with the intent to block the BMPRIa-CK2 interaction by binding to CK2 intracellularly, thereby keeping CK2 in circulation within the cell to maintain the downstream signaling as it does when the cell is bound to BMP-2.⁷⁴

In the first study,⁴³ the peptides were designed with the Antennapedia homeodomain signal sequence (QIKIWFQNRRKWKKMVPSDP) for cellular uptake. The authors evaluated the effect of the three CK2 peptides on chondrogenesis, and among these three peptide variants, only CK2.1 exhibited a chondrogenic potential. Specifically, CK2.1-stimulated C3H10T1/2 cells showed an increase in proteoglycan and collagen II synthesis equivalent to that of the BMP-2-positive control, as indicated by Alcian blue and immunostaining. Notably, immunostaining indicated a lower expression of collagen X and osteocalcin hypertrophy markers compared to BMP-2-treated cells. Interestingly, and in contrast, BMP-2, CK2.2, and CK2.3 induced mineralization in the C3H10T1/2 cells, but CK2.1 did not. Therefore, CK2.1 appeared to be selective for chondrogenesis, whereas CK2.2 and CK2.3 appeared to be selective for osteogenesis. It is unclear why this contrast among these three peptide variants was observed, given that all three were designed to operate by the same mechanism (i.e., inhibiting CK2 binding to BMPRIa).

For an in vivo evaluation with CK2.1, the authors then performed a systemic injection of CK2.1 into the tail vein of C57BL/6J mice, which resulted in enhanced articular cartilage formation around the femurs. Increased articular cartilage formation based on Safranin O (Saf-O)/fast green staining and collagen II and IX immunostaining was observed at levels equivalent to a BMP-2-positive control. Furthermore, an increase in expression of the hypertrophy marker collagen X was observed with BMP-2, but not with CK2.1.⁴³

In the second study, Akkiraju et al.⁴⁴ evaluated the performance of CK2.1 when conjugated to HA hydrogel particles (HGP) in a mouse intra-articular injection model. An induced OA-like condition was created by destabilization of the medial meniscus (DMM). Mice receiving intra-articular injections of HGP-CK2.1 following OA showed higher levels of collagen II and IX immunostaining along with low immunostaining of collagen X and osteocalcin compared to phosphate buffered saline (PBS) and HGP controls.⁴⁴

CK2.1 appears to be a promising and potentially chondroinductive peptide, although it was employed as a drug, as opposed to in a regenerative medicine context. That is, CK2.1 was delivered as a drug through intra-articular injection in an OA model, as opposed to being conjugated to a material to fill a cartilage defect. Therefore, future studies could perhaps investigate the performance of this CK2.1 peptide in a full articular cartilage defect model compared to a positive control to fully establish its chondroinductive potential. However, given that the proposed mechanism of action with CK2.1 is intracellular, it is unclear how CK2.1 might fare in such an approach.

HSNGLPL

HSNGLPL was discovered in 2010 by phage display as a peptide sequence with binding affinity to TGF-β1.⁴⁵ Shah et al.⁴⁵ engineered a self-assembling peptide amphiphile (PA) molecule, specifically a TGF binding PA (TGFBPA) that formed nanofibers with a high density of TGF-β1 binding epitopes exposed on the surface. The TGFBPA included the HSNGLPL sequence inside of the sequence of HSNGLPLGGGSEEEAAAVVV(K)-CO(CH₂)₁₀CH₃. These amphiphilic molecules assembled into hydrogels comprised an interconnected network of nanofibers in the presence of calcium chloride.

In vitro analysis with human mesenchymal stem cells (hMSCs; we assume from bone marrow) showed that one TGFBPA group exhibited higher aggrecan gene expression levels after 4 weeks of culture relative to a filler control (i.e., similar to TGFBPA, except without the HSNGLPLGGGS [the TGF binding domain] of the sequence), but only when cultured with TGF-β1, as no difference was identified between the TGFBPA groups and the filler control group in the absence of TGF-β1. Time points from 2 to 4 weeks for all groups containing TGF-β1 showed similar upregulation over time regardless of whether they were the TGFBPA groups or the filler PA group. As for glycosaminoglycan (GAG) production, there was no difference observed between the TGFBPA and filler PA groups in the presence of TGF-β1 after 3 weeks.

Moving in vivo with rabbits, 2 mm diameter, full-thickness chondral defects were created in the femoral trochlear groove and followed by MF. In this 12-week study, there were four groups: TGF-β1 alone, the filler PA hydrogel+TGF-β1, the TGFBPA hydrogel+TGF-β1, and the TGFBPA hydrogel alone. There was no sham (i.e., unfilled, MF only) control. Based on gross morphology, collagen II immunostaining, and GAG staining by Saf-O, it appeared the two groups with the TGFBPA hydrogel achieved superior regeneration. Notably, although there was no functional mechanical testing of regenerated cartilage, the TGFBPA hydrogel group achieved excellent structural regeneration without TGF-β1. In retrospect, a group of the filler PA hydrogel without TGF-β1 would have been a valuable point of comparison.⁴⁵

In 2018, Chen et al.⁴⁶ incorporated the HSNGLPL peptide at high concentrations into a porous chitosan sponge scaffold through a carbodiimide linker, and investigated the chondrogenic differentiation of porcine bone marrow-derived MSCs in vitro and in vivo. For the in vitro studies, the chitosan sponges were preloaded with TGF-β1 for 3 hours before culturing in TGF-β1-free medium. Scaffolds with the highest peptide concentration, that is, a chitosan to peptide mass ratio of 10:3, exhibited the highest gene expression levels of SOX9, collagen II, and aggrecan, even outperforming the positive control group (i.e., chitosan and TGF-β1 without peptide). In contrast, collagen X gene expression was not upregulated compared to the chitosan material negative control or positive control. It remains unknown how the HSNGLPL-conjugated chitosan scaffolds would have performed without the preloading of TGF-β1, which would be an appropriate comparison in future investigations.

In vivo, the authors evaluated the chitosan-HSNGLPL (10:3 mass ratio) scaffolds in a rabbit model with 4 mm diameter and 4 mm deep osteochondral defects in the femoral trochlear groove. There were three groups: a negative sham (i.e., empty defect) control, the chitosan scaffold alone, or the 10:3 chitosan-HSNGLPL scaffold. Cartilage regeneration was assessed after 3 and 6 months. The chitosan group alone appeared to have a detrimental effect compared to the sham control, and the addition of the peptide appeared to “rescue” the performance of the chitosan scaffold to put the regeneration more back on par with the negative control. The authors noted that the International Cartilage Regeneration & Joint Preservation Society (ICRS) scores were higher in the peptide group than in the negative sham control, but these results were not indicated to be statistically significant, perhaps due to the low sample number (i.e., n = 3) or the relative standard deviations. Nevertheless, the Saf-O staining showed a fairly consistent and continuous staining across the surface in the peptide group that was not as apparent in the negative control.

The intriguing question would be how the HSNGLPL peptide, which may have rescued the detrimental performance of chitosan alone, may fare under different circumstances such as conjugation to a different material, or if placed in a more weight-bearing region (e.g., femoral condyles), or if placed in a chondral-only defect instead of osteochondral.

In summary, the HSNGLPL peptide has shown some promise in vitro in conjugation to chitosan scaffolds, but required a preloading of TGF-β1 to achieve this effect and has shown some promise in vivo in rabbit trochlear groove defects as a self-assembling hydrogel (2 mm diameter chondral-only defects with MF) or by conjugation to chitosan sponges (4 mm diameter osteochondral defects).

However, further evaluation will be required before conclusions can be drawn for this TGF-β1 binding peptide. Specifically, the TGFBPA group was not compared to a filler PA control group, so it could not be determined whether the peptide itself was responsible for the quality of regeneration in the absence of TGF-β1. In the chitosan scaffold, the peptide may have helped to recover the detrimental performance of chitosan, but it remains to be seen whether the peptide in another biomaterial or defect type/location would significantly outperform a negative sham control. Moreover, functional mechanical testing (e.g., indentation stress relaxation) would be a valuable addition to future outcome analyses.⁷⁵ Further investigation of this intriguing peptide is warranted in cartilage regeneration.

His-Ala-Val

His-Ala-Val (HAV) is a conserved sequence in the first extracellular domain (ECD1) of N-cadherin, a transmembrane protein that plays a vital role in cell-cell interactions during mesenchymal condensation, a prerequisite to cartilage formation. Synthetic peptides containing the HAV domain have been shown to possess N-cadherin-like binding activity.^76,77

In 2013, Bian et al.⁴⁷ incorporated HAV (as HAVDIGGGC) into a methacrylated hyaluronic acid (MeHA) hydrogel and evaluated the role of the functionalized hydrogel in the chondrogenic differentiation of encapsulated human BMSCs (hBMSCs; we assume from bone marrow). In a 28-day culture period in chondrogenic medium containing TGF-β3, the gene expression of collagen II, aggrecan, and SOX9 in the cadherin peptide group on days 1 and 3 was significantly higher than the control and scrambled groups; however, by day 7, there were no significant difference among the groups. Following 28-day culture with hBMSCs, peptide-functionalized hydrogels possessed higher GAG and collagen content relative to the control and scrambled groups. The stand-alone chondroinductivity of HAV was not evaluated, as the medium for each group contained TGF-β3.

Moving in vivo with nude mice, the subcutaneous injection of HAV-functionalized hydrogels along with TGF-β3-loaded microspheres and hBMSCs resulted in a higher content of GAGs and collagen compared to the control and scrambled groups and more intense staining of collagen II and chondroitin sulfate (CS). The authors concluded that there was an enhancement of early chondrogenesis of BMSCs and cartilage-specific matrix production.⁴⁷ We emphasize that the stand-alone chondroinductivity of HAV was not evaluated, but instead its ability to enhance the chondrogenesis of TGF-β3 was evaluated, and indeed the data supported an enhancement.

In 2016, Vega et al.⁷⁸ used single-cell imaging to demonstrate that the incorporation of the HAV peptide augments the β-catenin recruitment to the cell membrane in hMSCs (we assume from bone marrow), followed by translocation to the nucleus.⁷⁸

In a follow-up study by the same group in 2018, Kwon et al.⁵³ investigated the effect of dosage and timing of the HAV peptides in HA hydrogels in the presence of TGF-β3 and showed that the effect of these N-cadherin peptides strongly depended on the dosage. Specifically, a dose-dependent increase in collagen II gene expression was observed during the first 7 days of culture; however, there were no significant differences among groups (including the peptide-free negative control) at 14 days. Following 56 days of culture, increased levels of GAGs and collagen II staining in HAV-containing groups were observed compared to unfunctionalized HA groups and scrambled peptide groups.⁵³ As highlighted by the authors, the chondroinductive potential of HAV alone without any additional growth factor is still an area to investigate, given that TGF-β3 was included with all groups.

In 2019, Eren Cimenci et al.⁵⁴ designed a self-assembling amphiphilic peptide nanofiber system containing the HAV peptide to induce the chondrogenic differentiation of rat BMSCs. The nanofiber systems' activity was evaluated by seeding rat BMSCs on HAV nanofibers, nonbioactive nanofibers, or uncoated tissue culture plates (TCP) in a commercially available chondrogenic medium. Saf-O staining and DMMB assay indicated that cells cultured on HAV nanofibers exhibited abundant GAG accumulation after culture for 14 days compared to cells cultured on nonbioactive nanofibers, and TCP. Gene expression analysis indicated an increase in collagen II, aggrecan, and SOX9 expression on days 3, 7, and 14 in cells cultured on HAV-nanofibers compared to cells cultured on nonbioactive nanofibers and TCP. The authors concluded that the HAV peptide and nanofiber system facilitated chondrogenesis.⁵⁴

In 2020, Feng et al.⁴⁸ fabricated an HAV-conjugated, aggrecanase-1 cleavable hydrogel and evaluated its regenerative potential in a rabbit osteochondral defect model. HAVDIGGGC peptide was conjugated into a hyperbranched poly(ethylene glycol) (PEG)-based multiacrylate polymer (HBPEG) and mixed with aggrecanase-1 cleavable peptide (ACpep) and cysteamine-modified CS to form (HAV-HBPEG)-CS-ACpep hydrogels. Rabbit BMSC-encapsulated hydrogels were evaluated in a rabbit model with 4 mm diameter and 4 mm deep osteochondral defects in the patellar groove. There were three groups: a negative sham control, the (HAV-HBPEG)-CS-ACpep with rabbit BMSCs, and HBPEG-CS-ACpep (with no HAV) with rabbit BMSCs. The repair potential of the hydrogels was evaluated after 12 and 18 weeks. Based on the gross morphological view, microcomputed tomography imaging, and histological analysis, the subchondral bone was partially repaired after 12 weeks and fully repaired after 18 weeks in all three groups, with higher bone volume and mineral content in the HAV-conjugated hydrogel group. As for the cartilage layer, immunostaining indicated that after 12 weeks, the HBPEG-CS-ACpep/BMSC hydrogel group exhibited more collagen II deposition than collagen I and more intense GAG staining by Saf-O, and periodic acid-Schiff (PAS) staining, compared to the sham and HAV-conjugated hydrogel group. Interestingly, after 18 weeks, the HAV-conjugated group exhibited intense Saf-O and PAS staining, indicating abundant deposition of GAGs and glycoproteins, in addition to intense collagen II immunostaining with no collagen I immunostaining compared to sham and HAV-free groups.⁴⁸ The presence of aggrecanase 1 cleavable peptide and HAV peptide appeared to have had a synergistic effect to enhance cartilage regeneration. It would have been interesting to evaluate the hydrogel's regenerative potential in the presence and absence of each of the components, that is, CS, HAV peptide, and aggrecanase 1 degradable peptide.

In 2021, Mohammed et al.⁷⁹ designed self-assembling hydrogels with tunable mechanical stiffness based on five different peptide sequences that mimicked the HAV motif with fluorenylmethyloxycarbonyl (Fmoc) as an aromatic assembling group into fibrillar structures. The five peptide sequences were Fmoc-GGHAV, Fmoc-GGHAVD, Fmoc-GGHAVS, Fmoc-GGHAVDI, and Fmoc-GGHAGDI. The chondroinductive potential of the soluble peptides and self-assembled peptide hydrogels was evaluated with hMSCs-3A6 cells. After 21 days in culture, the Fmoc-GGHAVDI peptide solution group exhibited the highest gene expression of collagen II compared to other peptide solutions and chondroinductive medium (containing TGF-β1). Aggrecan gene expression was equivalent between all peptide groups; SOX9 gene expression was mainly increased with chondroinductive media alone and with Fmoc-GGHAVDI. However, when hMSCs-3A6 cells were encapsulated in the peptide hydrogels, the Fmoc-GGHAVS with greater stiffness resulted in more intense Alcian blue staining indicating increased GAG deposition, and a significant increase in collagen II/collagen I ratio compared to other peptide hydrogels.

In 2021, Teng et al.⁴⁹ synthesized functionalized a MeHA hydrogel as a matrix for hBMSC chondrogenesis. Six hydrogel groups were synthesized: MeHA alone as a control (HA), MeHA+RGD (RHA), MeHA+HAV (HHA), MeHA+RGD+HAV (HR), HR loaded with kartogenin (K@HR), and HR hydrogel with KGN encapsulated in PLGA microspheres (K@PM-HR). Following 14 days of culture in chondrogenic medium, gene expression analysis indicated that the HR group significantly upregulated the gene expression of aggrecan, collagen II, and SOX9, compared to MeHA alone and MeHA functionalized with a single peptide. The kartogenin groups (K@HR and K@PM-HR) resulted in a significant increase in gene expression of aggrecan, collagen II, and SOX9. As for matrix synthesis, a significant increase in GAG content (DMMB assay) and collagen II content (hydroxyproline assay) was observed with all RGD functionalized groups compared to HA and HHA. At 28 days, HR hydrogels significantly upregulated the gene expression of aggrecan and collagen II compared to HA and HHA hydrogels. Interestingly, the K@PM-HR group resulted in the highest gene expression of aggrecan, collagen II, SOX9, and collagen X. The GAG and collagen contents in all RGD functionalized groups were significantly higher than HA and HHA groups.

Transitioning to in vivo, the subcutaneous injection of HA, HR, K@HR, or K@PM-HR in nude mice showed after 56 days that the GAG (DMMB assay) and collagen contents (hydroxyproline assay) were significantly higher in the kartogenin groups compared to the HA and HR groups, with the highest collagen content being for K@PM-HR. In addition, a significant increase in GAG and collagen content was observed with the HR group compared to the MeHA control group.

In summary, the HAV peptide was shown to be promising in vitro; however, it has been evaluated in the presence of TGF-β3 or TGF-β1, so it remains unknown whether the peptide alone is capable of chondroinduction. In vivo, the subcutaneous studies in nude mice suggested that the HAV peptide has enhanced chondroinduction in the presence of TGF-β3 or kartogenin. In the rabbit osteochondral defect study, the HAV peptide appeared to be promising when combined with aggrecanase 1 cleavable hydrogels. Additional evaluation will be required to determine whether the peptide alone or in combination with other biomaterials would be chondroinductive. Based on the studies performed so far, the major application of the HAV peptide is to facilitate chondrogenesis by enhancing cell-cell interactions.

Cytomodulins

Cytomodulins (CMs) are TGF-β1 mimicking peptides that have been reported to exhibit TGF-β1-like activity by enhancing the expression of collagen I and improving the wound healing effect of fibroblasts.⁸⁰ The original source of these “cytomodulins” can be traced back to patents from the late 1990s,^81,82 which perhaps were meant to mimic a β bend in the protein, as the sequences of LIANAK (a.k.a. cytomodulin 10, or CM-10) and ANVAENA (a.k.a. cytomodulin 1, or CM-1) do not actually appear in TGF-β.

In 2015, Zhang et al.⁵⁰ conjugated the CM-10 (LIANAK) peptide to functional nanofibrous hollow microspheres (FNF-HMS), which were leveraged as a delivery system for the CM-10 peptide. The microspheres were made from a poly(l-lactic acid) (PLLA)-based block copolymer. Three-week culture of CM-10-functionalized microspheres with rabbit BMSCs resulted in positive Saf-O staining, and the appearance of round cells encased in lacunae, suggesting chondrogenic differentiation of rabbit BMSCs compared to a material-only control and in the absence of additional growth factors. Moving then to a 2-week in vivo study, the subcutaneous injection of CM-10-functionalized microspheres in mice, along with rabbit BMSCs, resulted in ectopic cartilage-like tissue formation, as demonstrated by intense Saf-O staining of GAGs and collagen II immunostaining, with no apparent mineralization, compared to no cartilage formation in the material control.⁵⁰

In 2019, Park et al.⁵¹ used a click-crosslinked hyaluronic acid (Cx-HA) hydrogel as a scaffold, and physically loaded or covalently linked CM-10 to the hydrogel and tested the chondrogenic potential of both on human periodontal ligament stem cells (hPLSCs). In vitro culture of hPLSCs for 4 weeks with soluble CM-10 or TGF-β (type 1/2/3 not specified) chondrogenic medium showed increased staining of collagen II and GAGs compared to the peptide-free medium. Furthermore, hPLSCs cultured for 4 weeks with soluble CM-10 exhibited an increased gene expression of SOX9, aggrecan, and collagen II at a comparable fold increase to a positive control TGF-β group.

Transitioning to an in vivo study, hPLSCs and hydrogels were subcutaneously injected in mice, with three groups being the HA hydrogel alone, with CM physically entrapped, and with the CM conjugated. There was no TGF-β-positive control for this in vivo study. The CM was much more effective when conjugated than when included in soluble form. Specifically, increased staining of collagen II and GAGs and increased gene expression of SOX9, aggrecan, and collagen II were observed for the covalently linked CM group relative to the physically loaded CM and peptide-free hydrogels. Worth mentioning is that Park et al.⁵¹ refer to the peptide as CM-2; however, the sequence of the peptide provided in the article is LIANAK, which is that of CM-10, and CM-2 is LIAEAK as per Lam et al.⁸⁰ In summary, the TGF-β mimicking peptide CM-10 demonstrated upregulation of chondrogenic markers both in vitro and in vivo (subcutaneous injection in mice) relative to negative controls. However, comparison to a TGF-β-positive control was generally absent with the exception of one in vitro study that demonstrated chondrogenesis comparable to TGF-β. The CM-10 peptide may be a promising peptide that warrants investigation in an in vivo cartilage defect study.

B2A

B2A (a.k.a. B2A2-K-NS) is a synthetic multidomain peptide [(H-AISMLYLDENEKVVLKK(H-AISMLYLDENEKVVLK)-Ahx-Ahx-AhxRKRLDRIAR-NH2] that was recognized as a BMP-2 receptor modulator by Lin et al.⁵⁹ in 2005. The B2A design includes a heparin binding domain (RKRKLERIAR), a hydrophobic domain, and a receptor-targeted domain (AISMLYLDENEKVVL), with binding to both type I and II receptors, with selectivity for BMPR-Ib. B2A was found to enhance BMP-2 activity in vitro synergistically and was hypothesized that it might improve bone repair. Knowing the role of the BMP pathway in chondrogenesis, Lin et al.⁵² evaluated the chondrogenic potential of B2A peptide in vitro and in vivo. Polymerase chain reaction (PCR) array analysis of B2A-treated murine multipotential embryonic stem cell line C3H10T1/2 cells showed significant upregulation of fibroblast growth factor receptors 1 and 2 (FGFR1, FGFR2) and moderate upregulation of FGF1 genes. The upregulation of matrix genes (collagen I and II), and Smad1, Smad4, and Twist1 genes was additionally observed compared to the peptide-free group.

Furthermore, Alcian blue and collagen II staining indicated that B2A stimulated the production of GAGs and collagen II in hBMSCs and human chondrocytes (passage number not identified) in micromass culture. Transitioning to an in vivo rat model, the authors evaluated the activity of B2A by performing a pilot study in a chemically induced OA model. Following induction of OA, rats were injected intra-articularly with either saline or 500 ng of B2A. There appeared to be a significant repair of articular cartilage in the B2A-treated group versus saline group based on hematoxylin and eosin (H&E) and Saf-O staining.⁵²

In summary, it appears there may be some chondroinductive effect with B2A2 in vitro, and some chondroprotective effect in a rat OA model, but it remains to be seen whether B2A2 would elicit a chondroinductive effect in a cartilage defect model.

SPPEPS

In 2019, we identified the SPPEPS peptide as a similar sequence between two chondroinductive molecules, aggrecan and the TGF-β3 proprotein.⁵⁵ The chondroinductive potential of SPPEPS was assessed with rat BMSCs, and it was determined that the soluble peptide at 100 ng/mL increased the expression of collagen II compared to the negative control with negligible cytotoxic effects. In addition, proteomic analysis revealed that after 7 days in culture, the insulin signaling pathways were activated through the GSK-3β gene, which is involved in the maintenance of chondrocyte phenotype and cartilage ECM, in both SPPEPS and positive control groups. In addition, collagen II expression was increased when rat BMSCs were cultured on the surface of pentenoate-functionalized hyaluronic acid (PHA) hydrogels when a combination of both SPPEPS and the adhesion peptide RGD was provided, relative to either peptide alone. The SPPEPS peptide may be a promising peptide; however, additional studies are required to confirm its chondroinductive potential.⁵⁵

Link N (DHLSDNYTLDHDRAIH)

Link N peptide was first discovered in 1993 by Martin and Dean⁸³ by enzymatic cleavage of the link protein, and it was hypothesized that Link N might play a role in proteoglycan synthesis regulation. In 2003, Mwale et al.⁸⁴ reported that Link N peptide stimulated the production of proteoglycans and collagen II and IX in intervertebral disc (IVD) pellet-cultured cells. Similarly, Wang et al.⁸⁵ investigated the effect of Link N peptide in an ex vivo 3D culture of rabbit IVD cells. Real-time PCR, enzyme-linked immunosorbent assay, and Western blotting confirmed that Link N protein significantly upregulated the gene expression and synthesis of SOX9, aggrecan, and collagen II compared to a negative control and a scrambled peptide group. The authors showed that Link N peptide interacted with BMP-RII receptor and significantly increased the protein production of BMP-4 and BMP-7, but not BMP-2 or BMP-6.

In 2018, He et al.⁵⁶ studied the effects of Link N protein on the proliferation and chondrogenic differentiation of rat cartilage stem/progenitor cells (CSPCs). In vitro, the two-dimensional culture of CSPCs with increased concentrations of Link N protein (0–500 ng/mL) resulted in increased gene expression of SOX9, collagen II, and aggrecan, with no increase in Runx2 or collagen X expression compared to peptide-free groups. 3D pellet culture showed that Link N stimulated the chondrogenic differentiation of CSPCs, based on collagen II and SOX9 immunostaining; however, the best performance was for the peptide+TGF-β3 group compared to peptide-free and peptide groups.

GFOGER

The GFOGERGVEGPOGPA peptide was identified in 1998 as a sequence of residues 502–507 of the α1 collagen chain, located in the α1(I)CB3 fragment, based on its high binding affinity to α2β1 integrin.⁸⁶ In 2000, Knight et al.⁸⁷ determined that the actual recognition site is mainly in the sequence GFOGER and that this peptide sequence signifies a high-affinity binding site in collagen I and IV for α2β1 integrin, and in collagen I for α1β1 integrin.

In 2010, Liu et al.⁵⁷ incorporated a collagen mimetic peptide (CMP) containing GFOGER into a PEG hydrogel and evaluated its chondroinductive potential with encapsulated hBMSCs. In the presence of TGF-β3, peptide-containing hydrogels promoted the chondrogenesis of hBMSCs indicated by the enhanced staining of GAG, collagen II, and aggrecan compared to peptide-free hydrogels. Gene expression analysis showed an upregulation of SOX9 and downregulation of collagen X. Based on the in vitro data, the authors concluded that the presence of GFOGER induced chondrogenic activity in the presence of TGF-β3 and prevented or delayed hypertrophy.

In 2014, Mhanna et al.⁵⁸ incorporated GFOGER and matrix metalloproteinase (MMP)-sensitive motifs into PEG hydrogels, creating a functionalized degradable hydrogel, and compared the chondroinductive potential of this hydrogel with RGD-functionalized hydrogels and MMP-free (nondegradable) hydrogels. In the presence of TGF-β3, GFOGER-modified degradable gels provided the highest hBMSC proliferation rate, compared to peptide-free gels and RGD gels. GAG and DNA content were higher, but not statistically significant in the GFOGER-modified degradable gels compared to peptide-free and RGD hydrogels. Gene expression of collagen II was highest in GFOGER-modified degradable gels compared to peptide-free gels.

Worth mentioning is that the GFOGER was used as a PCL scaffold coating to enhance bone formation.⁸⁸ Reyes et al.⁸⁹ coated titanium surfaces with GFOGER to promote α2β1 integrin binding and found that the presence of GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells in an osteogenic medium, compared to peptide-free titanium. Recently, Clark et al.⁹⁰ found that GFOGER-functionalized hydrogels based on 4-arm PEG macromers with terminal maleimide group (PEG-4MAL) hydrogels prolonged hBMSC survival and improved bone repair in a mouse model.

In summary, GFOGER appears to elicit a favorable response, but since both bone and cartilage regeneration studies report favorable outcomes, the real question is how it may perform in vivo and whether osteogenesis versus chondrogenesis would be favored.

KIPKASSVPTELSAISTLYL

KIPKASSVPTELSAISTLYL was identified in 2003⁹¹ as a potential candidate to improve bone formation, and it represents residues 73–92 of BMP-2's knuckle epitope. In 2012, Renner et al.⁶⁰ evaluated the effect of KIPKASSVPTELSAISTLYL on hMSCs (we assume from bone marrow), and observed an increased production of GAGs relative to the negative control and at a value comparable to the BMP-2-positive control; however, when added with TGF-β3, this BMP-2-inspired peptide did not increase GAG production. A hydroxyproline assay showed a significant increase in total collagen with the KIPKASSVPTELSAISTLYL-treated cells, but at levels lower than the BMP-2-positive control. Interestingly, KIPKASSVPTELSAISTLYL did not increase the alkaline phosphatase (AP) activity or collagen I deposition compared to BMP-2-treated cells. Gene expression analysis showed an upregulation of aggrecan, COMP, and collagen II compared to the negative control, yet a reduced effect compared to BMP-2 and TGF-β3. In 2013, a follow-up article from the same group confirmed that KIPKASSVPTELSAISTLYL stimulated GAG production in hBMSCs to levels comparable to the positive control and in a full-factorial experiment, KIPKASSVPTELSAISTLYL stimulated GAG production without the need for other peptides or growth factors.⁶²

RYPISRPRKR and YKTNFRRYYRF

RYPISRPRKR (termed “HAbind” by the authors) is a peptide derived from the HA binding region of link protein. Link protein stabilizes the interaction between HA and the core protein of individual aggrecan molecules to form large aggrecan complexes in articular cartilage.^92,93 YKTNFRRYYRF (termed “CSbind”) was discovered by peptide array screening as a sequence that binds chondroitin-6-sulfate (C6S) and it was found to block the inhibitory activity of C6S on neurite outgrowth.⁹⁴

In 2015, Parmar et al.⁶¹ synthesized a biodegradable hydrogel from recombinant streptococcal collagen-like 2 (Scl2) proteins, functionalized with HAbind and CSbind peptides, and crosslinked with MMP7-sensitive peptide. The authors evaluated the chondrogenic differentiation of encapsulated hBMSCs, and in the presence of TGF-β3, both HAbind-MMP7-Scl2 and CSbind-MMP7-Scl2 hydrogels enhanced the gene expression of collagen II, aggrecan, and SOX9 in hBMSCs, with the highest gene upregulation of chondrogenic markers being for HAbind-MMP7-Scl2 at 10% functionalization compared to unfunctionalized hydrogels. Collagen I and X gene expression were significantly lower in HAbind-MMP7-Scl2 and CSbind-MMP7-Scl2 compared to unfunctionalized hydrogels. Furthermore, biochemical assays indicated that total collagen, GAG, and DNA content were highest for the HAbind (10%)-MMP7-Scl2 hydrogels compared to all other groups.

GRVDWLQRNANFYDWFVAELG (Insulin Peptide)

GRVDWLQRNANFYDWFVAELG peptide was created by phage display and recognized by its affinity for insulin receptor and thought to mimic insulin-like growth factor (IGF)-1.⁹⁵ In 2013, Renner and Liu⁶² investigated the chondrogenic effect of the insulin peptide in soluble form on human mesenchymal stem cells (MSCs; we assume from bone marrow). In the presence of insulin and TGF-β3, the insulin peptide treatment resulted in an increased production of GAG compared to the TGF-β3-positive control. Interestingly, the insulin peptide was combined with the TGF-β1-inspired peptide (ANVAENA) to mimic the synergy observed between the insulin peptide and full-length TGF-β3 protein; however, the presence of both peptides resulted in a decrease in GAG production compared to having one peptide only. The authors hypothesized that this effect might have been due to a change in experimental conditions (culture time, passage number, and rBMSC expansion conditions), or it may be that the TGF-β1 peptide acted on different pathways than TGF-β3.

Other Peptides

Several peptides have been reported to be used for cartilage regeneration, which are summarized in Tables 3 and 4 (in vivo studies) and 5 (in vitro studies). Among these peptides are self-assembling peptides such as KLD^{66,67,73,96–98} and RADA^68,73 (commercially available as PuraMatrix^™), which may provide chondro-supportive scaffolds, but were not designed specifically for chondroinduction. In vivo evaluation of KLD peptide in a full-thickness cartilage defect rabbit model⁶⁶ showed that the KLD hydrogel alone resulted in significantly more intense Saf-O staining and collagen type II immunostaining compared to KLD hydrogels containing chondrogenic factors or BMSCs. However, a later study performed in a 15 mm diameter cartilage defect equine model⁶⁷ showed that KLD treatment improved the clinical outcome and filling. However, there were decreased levels of aggrecan and collagen type II, resulting in a poor repair tissue quality compared to MF.⁶⁷

E7 peptide (EPLQLKM), identified in 2012,⁹⁹ is a strong MSC affinity peptide candidate. A biphasic scaffold made of demineralized bone matrix (DBM) and chitosan (CS) hydrogel was functionalized with E7 and implanted in an osteochondral defect rabbit model in combination with MF.⁶⁴ The E7-functionalized scaffolds resulted in superior cartilage regeneration with no sign of hypertrophy relative to MF and unfunctionalized control scaffolds.⁶⁴ In another study by the same group,⁶⁵ DBM particles were functionalized with E7 and combined with CS hydrogel (DBM-E7/CS). DBM-E7/CS scaffold implanted into the fossa iliaca subcutaneous region of athymic nude mice for 4 weeks resulted in the formation of a translucent cartilage-like tissue superior to that generated by material control groups (i.e., CS and DBM/CS).

WYRGRL, a collagen II affinity peptide, and KLER, a decorin-derived peptide, were evaluated for their chondroinductive potential in vitro and in vivo through subcutaneous injection in a mouse model.⁶³ These peptides were not found to be attractive for collagen II deposition. KLER was additionally assessed in 2009⁷⁰ and was found to promote chondrogenesis of hBMSCs in chondrogenic medium (+TGF-β1) when combined with RGD in a PEG scaffold, compared to control medium and scrambled peptide group. However, no chondroinductive activity was observed in the absence of TGF-β1.

ANVAENA, like CM-10 (reviewed above) is a TGF-β1-inspired peptide. ANVAENA was evaluated for its chondroinductive potential with hBMSCs in its soluble form, but unlike CM-10, it was found to decrease the synthesis of GAGs compared to a negative control.⁶²

In addition, GTPGPQGIAGQRGVV (termed P15 by the authors) is a collagen I-inspired peptide that was found to enhance the commitment of C3H10T1/2 cells toward the chondrogenic lineage in the presence of TGF-β.⁷¹ GPPDWHWKAMTH peptide (termed R1-P1 by the authors) was inspired from FGFR1 and was found to exhibit chondroprotective effects.⁶⁹ CDPGYIGSR-modified scaffolds inspired by laminin supported the proliferation of bovine knee chondrocytes (BKCs) and increased GAG and collagen content.⁷² Interestingly, a lot of progress has been made in the past few years with several laminin-mimetic peptides, including YIGSR, to modulate the behavior of the nucleus pulposus (NP) of the IVD in terms of cell attachment, morphology, signaling, and phenotype.^100–102 However, to the best of our knowledge, no additional study reported the chondroinductive potential of these peptides in vitro with BMSCs or in vivo in a cartilage defect model in the absence of exogenous growth factors.

In addition to the previously discussed peptides, we highlight two additional peptides, TPX-100 and Engedi1000, which are currently in clinical trials as DMOADs. TPX-100 is a 23-amino acid peptide derived from matrix extracellular phosphoglycoprotein (MEPE) that has been evaluated in clinical studies as a DMOAD.^103–105 Based on a published abstract,¹⁰⁶ intra-articular injections of TPX-100 in goats resulted in hyaline cartilage formation. The abstract mentioned that based on the performed clinical trials, TPX-100 injections were safe and well tolerated, and improved knee function.¹⁰⁶

Engedi1000 (E1K) is a synthetic peptide from Ensol Biosciences, Inc. (Daejeon, South Korea), which is currently in phase 1 clinical studies at Seoul National University Hospital.¹⁰⁷ The manufacturers claim that E1K blocks the Smad1/5/8 pathway, which promotes degeneration of cartilage tissue, through blocking TGF-β1, and maintains the Smad2/3 pathway that induces cartilage tissue regeneration. However, we have been unable to locate peer-reviewed publications evaluating the activity of TPX-100 and E1K peptides in full cartilage defect models.

Discussion

Among the peptides discovered to date, there exists great potential for synthetic peptides to induce the chondrogenic differentiation of stem cells. It is no surprise that the search for chondroinductive peptides has commonly been inspired by proteins that are known to play a role in chondrogenesis. TGF-β1 and TGF-β3 are widely used to induce chondrogenesis in vitro and are the most targeted growth factors with four peptides reported: HSNGLPL, CM-10, and ANVAENA from TGF-β1 and SPPEPS from both TGF-β3 and aggrecan. HSNGLPL, which is a TGF-β1 affinity peptide, seems promising based on the in vivo cartilage studies done in the absence of any exogenous growth factor. CM-10 looks promising as well; however, it has not yet been tested in a full articular cartilage defect model in vivo. ANVAENA did not appear to be chondroinductive based on the studies done so far.

BMP-2 is known to induce chondrogenesis and osteogenesis, and there are three peptides reported to be inspired by BMP-2, which are B2A, KIPKASSVPTELSAISTLYL, and CK2.1. Both B2A and KIPKASSVPTELSAISTLYL were initially identified for their ability to enhance bone formation, and their chondrogenic potential was later assessed. While there are no reported chondroinductive studies done in vivo for KIPKASSVPTELSAISTLYL, this peptide did show an advantage in comparison to BMP-2 in vitro as it did not lead to an increase of AP activity or collagen I synthesis. B2A was tested in vivo in a chemically induced OA model; however, there are no reported studies in a full osteochondral defect model so far. CK2.1 is the most recently investigated peptide, and compared to BMP-2, this peptide enhanced articular cartilage formation with no increase in collagen X expression following injection; however, CK2.1 assessment in vivo was done in a DMM model only. Additional investigation is necessary to fully identify the regenerative potential of these BMP-2-inspired peptides and to decipher their mechanisms of action.

Overall, there are a limited number of in vivo studies, and predominantly these are studies where the peptide was delivered by injection, systemically, intra-articularly, or subcutaneously. Of the peptides designed to be chondroinductive, only two have been implanted in a cartilage defect, which are HSNGLPL and HAVDI (Fig. 2). The call to action for the regenerative medicine community is therefore to evaluate chondroinductive peptides in well-controlled cartilage defect studies in vivo.

FIG. 2.

Categories of chondroinductive peptides that have been evaluated for cartilage regeneration without the addition of growth factors. Example peptides are grouped based on their parent molecules or pathways. Peptides that have been evaluated in vivo are highlighted in green and blue. Peptides highlighted in green have to date been evaluated only subcutaneously or through intra-articular injections. Peptides highlighted in blue with a dashed outline were evaluated in cartilage defect models. Citations to individual peptides may be found in Tables 1 and 2. GRVDWL… = GRVDWLQRNANFYDWFVAELG; KIPKASS… = KIPKASSVPTELSAISTLYL. Color images are available online.

While significant progress has been made in terms of cell therapy, scaffolds, and growth factors in the regenerative medicine community, pharmaceutical companies have been in a race to provide the first human use-approved DMOAD. However, the ultimate breakthrough that can change the current standard of care (microdrilling) would be to develop a cost-effective synthetic biomaterial that would regenerate true hyaline cartilage without the addition of any exogenous cell or growth factor. Having clinical translation in mind, such a biomaterial will be advantageous in terms of safety, cost, and regulatory approvals. Furthermore, the discovery of synthetic chondroinductive materials would potentially enhance currently existing treatments (e.g., MACI and MF) or set the stage for a completely new treatment approach.

Synthetic chondroinductive agents are mainly small molecules or peptides. Small molecules are mainly evaluated as DMOADs and are usually administered through intra-articular injections, which can range from one to several injections over the course of several weeks depending on the drug, which might be a limiting factor. On the other hand, peptides evaluated for cartilage regeneration fall into two categories. In the first category are peptides that are not designed to induce chondrogenesis, and in the second category are peptides that are designed to induce chondrogenesis. Category 1 includes peptides that have yet to exhibit evidence of chondroinduction in the absence of a growth factor (e.g., WYRGRL, KLER, and ANVAENA) and potential chondroprotective peptides (e.g., CDPGYIGSR). The first category additionally comprises promising self-assembling peptides that are themselves scaffolds (e.g., RADA and KLD) or MSC affinity peptides (e.g., E7). In vitro studies in this category did not include a positive control (except ANVAENA), and most of them relied on TGF-β to induce chondrogenesis. As for in vivo studies, only E7, RADA, and KLD were evaluated in a full-thickness cartilage defect, but no positive control was included in those studies.

The second category includes peptides focused on chondrogenesis; most peptides were evaluated in vitro only and most had TGF-β added to the culture medium (e.g., HSNGLPL, GFOGER, RYPISRPRKR, YKTNFRRYYRF, and GRVDWLQRNANFYDWFVAELG) or lacked a representative positive control (e.g., SPPEPS, CK2.1, B2A). In vivo, and for the exception of HSNGLPL and HAVDI, all remaining peptides (i.e., CK2.1, CM-10, and B2A) were evaluated through subcutaneous or intra-articular injection. Therefore, while these peptides seem promising, performing additional well-controlled cartilage defect studies in vivo is necessary to assess their chondroinductive potential.

Peptides represent promising synthetic chondroinductive agents that can be reproducibly and inexpensively produced and would allow the design of a synthetic chondroinductive material that can induce true hyaline cartilage regeneration. The main target for synthetic agents in cartilage regeneration is to mimic the protein-protein interactions between growth factors, cells, and/or ECM components. Bearing the common wide surface area of the protein-protein interactions, peptides are more likely to achieve this goal by covering the surface area with high specificity. Such specificity reduces the side effects of the peptide therapeutics, as they are not likely to bind to the proteins other than their targets, compared to small drugs with promiscuous binding affinity.

Peptides are advantageous in biocompatibility, as they are biodegradable into amino acids, and do not accumulate in the body.¹⁰⁸ In addition, peptides can be easily modified with different functional groups without complex chemistry, and various peptide chains can be combined such as adding a cell-penetrating sequence to a chondrogenic peptide sequence to enable intracellular delivery. Peptides can be conjugated to natural and synthetic polymers to form ECM-like scaffolds to trigger pathways associated with chondroinduction by binding to the appropriate surface receptor. Importantly, peptides allow the synthesis of a biomaterial device that is itself chondroinductive. Engineering of peptide-based therapeutics provides new and exciting opportunities that alternative chondroinductive agents cannot provide.

Conclusion

Chondroinductive peptides are a promising tool to design and engineer scalable synthetic chondroinductive biomaterials. We recommend further investigation of such peptide-modified biomaterials in well-designed cartilage defect models without the addition of any growth factor to evaluate chondroinduction in vivo. The cell biology of hyaline cartilage chondrocytes involves several interconnected prochondrogenic and antichondrogenic signaling pathways that interplay to maintain the hyaline phenotype; therefore, several peptide candidates are yet to be evaluated and synergistic activities among different peptides are yet to be investigated. We additionally advocate for the continued discovery of new peptides, and for rigorously designed in vivo studies with appropriate positive and negative controls to evaluate their efficacy.

Footnotes

Acknowledgment

We gratefully acknowledge the University of Oklahoma Gallogly College of Engineering PhD Recruitment Excellence Fellowship (PREF).

Authors' Contributions

M.S.D. contributed to the vision and conception of the article and B.A. drafted the original article. H.A. contributed to peptide-related content and provided general edits. M.S.D., B.A., and H.A. approve this version.

Disclosure Statement

No competing financial interests exist.

Funding Information

We gratefully acknowledge support from the Stephenson Graduate Fellowship (to B.A.).

References

United States Bone and Joint

Initiative

. The Burden of Musculoskeletal Diseases in the United States (BMUS): Prevalence, Societal and Economic Cost (4th Edition). https://www.boneandjointburden.org (accessed August 30, 2021).

Jones

I.A.

, Togashi

, Wilson

M.L.

, Heckmann

, and Vangsness

C.T.

Intra-articular treatment options for knee osteoarthritis. Nat Rev Rheumatol, 15, 77, 2019.

Steadman

J.R.

, Rodkey

W.G.

, and Briggs

K.K.

Microfracture: its history and experience of the developing surgeon. Cartilage, 1, 78, 2010.

Medvedeva

E.V.

, Grebenik

E.A.

, Gornostaeva

S.N.

, et al. Repair of damaged articular cartilage: current approaches and future directions. Int J Mol Sci, 19, 2366, 2018.

Richter

D.L.

, Schenck

R.C.

, Wascher

D.C.

, and Treme

Knee articular cartilage repair and restoration techniques. Sports Health, 8, 153, 2016.

Brittberg

, Lindahl

, Nilsson

, Ohlsson

, Isaksson

, and Peterson

Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 331, 889, 1994.

Brittberg

Clinical articular cartilage repair—an up to date review. Ann Joint, 3, 8, 2018.

Davies

R.L.

, and Kuiper

N.J.

Regenerative medicine: a review of the evolution of autologous chondrocyte implantation (ACI) therapy. Bioengineering (Basel), 6, 22, 2019.

U.S. Food and Drug Administration. FDA approves first autologous cellularized scaffold for the repair of cartilage defects of the knee. https://www.fda.gov/news-events/press-announcements/fda-approves-first-autologous-cellularized-scaffold-repair-cartilage-defects-knee (accessed August 30, 2021).

10.

, Shi

, Liu

, et al. Is implantation of autologous chondrocytes superior to microfracture for articular-cartilage defects of the knee? A systematic review of 5-year follow-up data. Int J Surg, 68, 56, 2019.

11.

, Chen

, Xu

, et al. Advances of injectable hydrogel-based scaffolds for cartilage regeneration. Regen Biomater, 6, 129, 2019.

12.

Słynarski

, de Jong

W.C.

, Snow

, Hendriks

J.A.A.

, Wilson

C.E.

, and Verdonk

Single-stage autologous chondrocyte-based treatment for the repair of knee cartilage lesions: two-year follow-up of a prospective single-arm multicenter study. Am J Sports Med, 48, 1327, 2020.

13.

Arshi

, Petrigliano

F.A.

, Williams

R.J.

, and Jones

K.J.

Stem cell treatment for knee articular cartilage defects and osteoarthritis. Curr Rev Musculoskelet Med, 13, 20, 2020.

14.

Zhang

, Hu

, Liu

, et al. Articular cartilage regeneration: the role of endogenous mesenchymal stem/progenitor cell recruitment and migration. Semin Arthritis Rheum, 50, 198, 2020.

15.

Doyle

E.C.

, Wragg

N.M.

, and Wilson

S.L.

Intraarticular injection of bone marrow-derived mesenchymal stem cells enhances regeneration in knee osteoarthritis. Knee Surg Sports Traumatol Arthrosc, 28, 3827, 2020.

16.

Armiento

A.R.

, Stoddart

M.J.

, Alini

, and Eglin

Biomaterials for articular cartilage tissue engineering: learning from biology. Acta Biomater, 65, 1, 2018.

17.

Roseti

, Desando

, Cavallo

, Petretta

, and Grigolo

Articular cartilage regeneration in osteoarthritis. Cells, 8, 1305, 2019.

18.

Jessop

Z.M.

, Manivannan

, Zhang

, Thornton

C.A.

, Narayan

, and Whitaker

I.S.

Tissue specific stem/progenitor cells for cartilage tissue engineering: a systematic review of the literature. Appl Phys Rev, 6, 031301, 2019.

19.

Graceffa

, Vinatier

, Guicheux

, Stoddart

, Alini

, and Zeugolis

D.I.

Chasing chimeras—the elusive stable chondrogenic phenotype. Biomaterials, 192, 199, 2019.

20.

, Zhang

, Romain

, Mak

, and Khan

Synovium-derived mesenchymal stem cell transplantation in cartilage regeneration: a PRISMA review of in vivo studies. Front Bioeng Biotechnol, 7, 314, 2019.

21.

Francis

S.L.

, Di Bella

, Wallace

G.G.

, and Choong

P.F.M.

Cartilage tissue engineering using stem cells and bioprinting technology—barriers to clinical translation. Front Surg, 5, 70, 2018.

22.

Lo Monaco

, Merckx

, Ratajczak

, et al. Stem cells for cartilage repair: preclinical studies and insights in translational animal models and outcome measures. Stem Cells Int, 2018, 9079538, 2018.

23.

Kwan

, Chisari

, and Khan

W.S.

Cell-free scaffolds as a monotherapy for focal chondral knee defects. Materials (Basel), 13, 306, 2020.

24.

Eftekhari

, Maleki Dizaj

, Sharifi

, et al. The use of nanomaterials in tissue engineering for cartilage regeneration; current approaches and future perspectives. Int J Mol Sci, 21, 536, 2020.

25.

Del Bakhshayesh

A.R.

, Asadi

, Alihemmati

, et al. An overview of advanced biocompatible and biomimetic materials for creation of replacement structures in the musculoskeletal systems: focusing on cartilage tissue engineering. J Biol Eng, 13, 85, 2019.

26.

Koh

R.H.

, Jin

, Kim

, and Hwang

N.S.

Inflammation-modulating hydrogels for osteoarthritis cartilage tissue engineering. Cells, 9, 419, 2020.

27.

Bao

, Li

, Yang

, et al. Advancements and frontiers in the high performance of natural hydrogels for cartilage tissue engineering. Front Chem, 8, 53, 2020.

28.

Walker

, Luo

, Pringle

E.W.

, and Cantini

ChondroGELesis: hydrogels to harness the chondrogenic potential of stem cells. Mater Sci Eng C Mater Biol Appl, 121, 111822, 2021.

29.

Morelli

, Bourgeas

, and Roche

Chemical and structural lessons from recent successes in protein-protein interaction inhibition (2P2I). Curr Opin Chem Biol, 15, 475, 2011.

30.

Cesa

L.C.

, Mapp

A.K.

, and Gestwicki

J.E.

Direct and propagated effects of small molecules on protein–protein interaction networks. Front Bioeng Biotechnol, 3, 119, 2015.

31.

Acar

, Ting

J.M.

, Srivastava

, LaBelle

J.L.

, and Tirrell

M.V.

Molecular engineering solutions for therapeutic peptide delivery. Chem Soc Rev, 46, 6553, 2017.

32.

, Liu

, Chen

, Lou

, Jiang

, and Zhang

Small molecule compounds promote the proliferation of chondrocytes and chondrogenic differentiation of stem cells in cartilage tissue engineering. Biomed Pharmacother, 131, 110652, 2020.

33.

Johnson

K.A.

, Woods

A.K.

, Joseph

S.B.

, et al. Development of KA34 as a cartilage regenerative therapy for osteoarthritis. Osteoarthritis Cartilage, 28, S518, 2020.

34.

Deshmukh

, Hu

, Barroga

, et al. A small-molecule inhibitor of the Wnt pathway (SM04690) as a potential disease modifying agent for the treatment of osteoarthritis of the knee. Osteoarthritis Cartilage, 26, 18, 2018.

35.

Deshmukh

, O'Green

A.L.

, Bossard

, et al. Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment. Osteoarthritis Cartilage, 27, 1347, 2019.

36.

Yazici

, McAlindon

T.E.

, Fleischmann

, et al. A novel Wnt pathway inhibitor, SM04690, for the treatment of moderate to severe osteoarthritis of the knee: results of a 24-week, randomized, controlled, phase 1 study. Osteoarthritis Cartilage, 25, 1598, 2017.

37.

Yazici

, McAlindon

T.E.

, Gibofsky

, et al. Results from a 52-week randomized, double-blind, placebo-controlled, phase 2 study of a novel, intra-articular wnt pathway inhibitor (SM04690) for the treatment of knee osteoarthritis. Osteoarthritis Cartilage, 26, S293, 2018.

38.

Yazici

, McAlindon

T.E.

, Gibofsky

, et al. Lorecivivint, a novel intraarticular CDC-like kinase 2 and dual-specificity tyrosine phosphorylation-regulated kinase 1A inhibitor and Wnt pathway modulator for the treatment of knee osteoarthritis: a phase II randomized trial. Arthritis Rheumatol, 72, 1694, 2020.

39.

Samumed

LLC

. A phase 3, 28-week, multicenter, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of a single injection of

SM04690 injected in the target knee joint of moderately to severely symptomatic osteoarthritis subjects

. clinicaltrials. gov

July 2020

. Report

. NCT04385303. https://clinicaltrials.gov/ct2/show/NCT04385303 (accessed August 30, 2021).

40.

Liu

, Jia

, Duan

, Xiong

, Wang

, and Ding

Functional peptides for cartilage repair and regeneration. Am J Transl Res, 10, 501, 2018.

41.

Gonzalez-Fernandez

, Sikorski

, and Leach

J.K.

Bio-instructive materials for musculoskeletal regeneration. Acta Biomater, 96, 20, 2019.

42.

Mahzoon

, and Detamore

M.S.

Chondroinductive peptides: drawing inspirations from cell-matrix interactions. Tissue Eng Part B Rev, 25, 249, 2019.

43.

Akkiraju

, Bonor

, and Nohe

CK2.1, a novel peptide, induces articular cartilage formation in vivo. J Orthop Res, 35, 876, 2017.

44.

Akkiraju

, Srinivasan

P.P.

, Xu

, Jia

, Safran

C.B.K.

, and Nohe

CK2.1, a bone morphogenetic protein receptor type Ia mimetic peptide, repairs cartilage in mice with destabilized medial meniscus. Stem Cell Res Ther, 8, 82, 2017.

45.

Shah

R.N.

, Shah

N.A.

, Lim

M.M.D

.R., Hsieh

, Nuber

, and Stupp

S.I.

Supramolecular design of self-assembling nanofibers for cartilage regeneration. Proc Natl Acad Sci U S A, 107, 3293, 2010.

46.

Chen

, Li

, Wang

, et al. TGF-β1 affinity peptides incorporated within a chitosan sponge scaffold can significantly enhance cartilage regeneration. J Mater Chem B, 6, 675, 2018.

47.

Bian

, Guvendiren

, Mauck

R.L.

, and Burdick

J.A.

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis. Proc Natl Acad Sci U S A, 110, 10117, 2013.

48.

Feng

, Zhou

, Xu

, Ye

, Gou

, and Gao

Enhanced regeneration of osteochondral defects by using an aggrecanase-1 responsively degradable and N-cadherin mimetic peptide-conjugated hydrogel loaded with BMSCs. Biomater Sci, 8, 2212, 2020.

49.

Teng

, Zhang

, Pan

, et al. A chondrogenesis induction system based on a functionalized hyaluronic acid hydrogel sequentially promoting hMSC proliferation, condensation, differentiation, and matrix deposition. Acta Biomater, 122, 145, 2021.

50.

Zhang

, Gupte

M.J.

, Jin

, and Ma

P.X.

Injectable peptide decorated functional nanofibrous hollow microspheres to direct stem cell differentiation and tissue regeneration. Adv Funct Mater, 25, 350, 2015.

51.

Park

S.H.

, Seo

J.Y.

, Park

J.Y.

, et al. An injectable, click-crosslinked, cytomodulin-modified hyaluronic acid hydrogel for cartilage tissue engineering. NPG Asia Mater, 11, 1, 2019.

52.

Lin

, Shanmugasundaram

, Liu

, Derrien

, Nurminskaya

, and Zamora

P.O.

B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats. J Orthop Res, 30, 1221, 2012.

53.

Kwon

M.Y.

, Vega

S.L.

, Gramlich

W.M.

, Kim

, Mauck

R.L.

, and Burdick

J.A.

Dose and timing of N-cadherin mimetic peptides regulate MSC chondrogenesis within hydrogels. Adv Healthc Mater, 7, 1701199, 2018.

54.

Eren Cimenci

, Kurtulus

G.U.

, Caliskan

O.S.

, Guler

M.O.

, and Tekinay

A.B.

N-cadherin mimetic peptide nanofiber system induces chondrogenic differentiation of mesenchymal stem cells. Bioconjugate Chem, 30, 2417, 2019.

55.

Mahzoon

, Townsend

J.M.

, Lam

T.N.

, Sjoelund

, and Detamore

M.S.

Effects of a bioactive SPPEPS peptide on chondrogenic differentiation of mesenchymal stem cells. Ann Biomed Eng, 47, 2308, 2019.

56.

, Wang

, Cui

, et al. Link protein N-terminal peptide as a potential stimulating factor for stem cell-based cartilage regeneration. Stem Cells Int, 2018, 3217895, 2018.

57.

Liu

S.Q.

, Tian

, Hedrick

J.L.

, Po Hui

J.H.

, Rachel Ee

P.L.

, and Yang

Y.Y.

Biomimetic hydrogels for chondrogenic differentiation of human mesenchymal stem cells to neocartilage. Biomaterials, 31, 7298, 2010.

58.

Mhanna

, Öztürk

, Vallmajo-Martin

, Millan

, Müller

, and Zenobi-Wong

GFOGER-modified MMP-sensitive polyethylene glycol hydrogels induce chondrogenic differentiation of human mesenchymal stem cells. Tissue Eng Part A, 20, 1165, 2014.

59.

Lin

, Zamora

P.O.

, Albright

, Glass

J.D.

, and Peña

L.A.

Multidomain synthetic peptide B2A2 synergistically enhances BMP-2 in vitro. J Bone Miner Res, 20, 693, 2005.

60.

Renner

J.N.

, Kim

, and Liu

J.C.

Bone morphogenetic protein-derived peptide promotes chondrogenic differentiation of human mesenchymal stem cells. Tissue Eng Part A, 18, 2581, 2012.

61.

Parmar

P.A.

, Chow

L.W.

, St-Pierre

J.-P.

, et al. Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration. Biomaterials, 54, 213, 2015.

62.

Renner

J.N.

, and Liu

J.C.

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments. Biotechnol Prog, 29, 1550, 2013.

63.

Hesse

, Freudenberg

, Niemietz

, et al. Peptide-functionalized starPEG/heparin hydrogels direct mitogenicity, cell morphology and cartilage matrix distribution in vitro and in vivo. J Tissue Eng Regen Med, 12, 229, 2018.

64.

Huang

, Zhang

, Hu

, et al. A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration. Biomaterials, 35, 9608, 2014.

65.

Meng

, Man

, Dai

, et al. A composite scaffold of MSC affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration. Sci Rep, 5, 17802, 2015.

66.

Miller

R.E.

, Grodzinsky

A.J.

, Vanderploeg

E.J.

, et al. Effect of self-assembling peptide, chondrogenic factors, and bone marrow derived stromal cells on osteochondral repair. Osteoarthritis Cartilage, 18, 1608, 2010.

67.

Miller

R.E.

, Grodzinsky

A.J.

, Barrett

M.F.

, et al. Effects of the combination of microfracture and self-assembling peptide filling on the repair of a clinically relevant trochlear defect in an equine model. J Bone Joint Surg Am, 96, 1601, 2014.

68.

, Shen

, Sun

, et al. Increased recruitment of endogenous stem cells and chondrogenic differentiation by a composite scaffold containing bone marrow homing peptide for cartilage regeneration. Theranostics, 8, 5039, 2018.

69.

Tan

, Chen

, Wang

, et al. A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice. Osteoarthritis Cartilage, 26, 1733, 2018.

70.

Salinas

C.N.

, and Anseth

K.S.

Decorin moieties tethered into PEG networks induce chondrogenesis of human mesenchymal stem cells. J Biomed Mater Res A, 90A, 456, 2009.

71.

Zhang

, Eisenhauer

, Kaya

, et al. P15 peptide stimulates chondrogenic commitment and endochondral ossification. Int Orthop, 41, 1413, 2017.

72.

Kuo

Y.-C.

, and Wang

C.-C.

Surface modification with peptide for enhancing chondrocyte adhesion and cartilage regeneration in porous scaffolds. Colloids Surf B Biointerfaces, 84, 63, 2011.

73.

Kopesky

P.W.

, Vanderploeg

E.J.

, Sandy

J.S.

, Kurz

, and Grodzinsky

A.J.

Self-assembling peptide hydrogels modulate in vitro chondrogenesis of bovine bone marrow stromal cells. Tissue Eng Part A, 16, 465, 2010.

74.

Bragdon

, Thinakaran

, Moseychuk

, et al. Casein kinase 2 β-subunit is a regulator of bone morphogenetic protein 2 signaling. Biophys J, 99, 897, 2010.

75.

Patel

J.M.

, Wise

B.C.

, Bonnevie

E.D.

, and Mauck

R.L.

A systematic review and guide to mechanical testing for articular cartilage tissue engineering. Tissue Eng Part C Methods, 25, 593, 2019.

76.

Blaschuk

O.W.

, Sullivan

, David

, and Pouliot

Identification of a cadherin cell adhesion recognition sequence. Dev Biol, 139, 227, 1990.

77.

Williams

, Williams

, Gour

B.J.

, Blaschuk

O.W.

, and Doherty

A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif. J Biol Chem, 275, 4007, 2000.

78.

Vega

S.L.

, Kwon

, Mauck

R.L.

, and Burdick

J.A.

Single cell imaging to probe mesenchymal stem cell N-cadherin mediated signaling within hydrogels. Ann Biomed Eng, 44, 1921, 2016.

79.

Mohammed

, Lai

T.-S.

, and Lin

H.-C.

Substrate stiffness and sequence dependent bioactive peptide hydrogels influence the chondrogenic differentiation of human mesenchymal stem cells. J Mater Chem B, 9, 1676, 2021.

80.

Lam

H.J.

, Li

, Lou

, Chu

, and Bhatnagar

R.S.

Synthetic peptides cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2) promote collagen synthesis and wound healing in vitro. Conf Proc IEEE Eng Med Biol Soc, 2004, 5028, 2004.

81.

Bhatnagar

R.S.

, and Qian

J.J.

Peptide compositions with growth factor-like activity. 1997. https://patents.google.com/patent/US5661127A/en?oq=%235%2c661%2c127 (accessed February 15, 2021).

82.

Bhatnagar

R.S.

, and Qian

J.J.

Peptide compositions with growth factor-like activity. 1998. https://patents.google.com/patent/US5780436A/en (accessed February 15, 2021).

83.

Martin

, and Dean

An N-terminal peptide from link protein is rapidly degraded by chondrocytes, monocytes and B cells. Eur J Biochem, 212, 87, 1993.

84.

Mwale

, Demers

C.N.

, Petit

, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem, 88, 1202, 2003.

85.

Wang

, Weitzmann

M.N.

, Sangadala

, Hutton

W.C.

, and Yoon

S.T.

Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J Biol Chem, 288, 28243, 2013.

86.

Knight

C.G.

, Morton

L.F.

, Onley

D.J.

, et al. Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence. J Biol Chem, 273, 33287, 1998.

87.

Knight

C.G.

, Morton

L.F.

, Peachey

A.R.

, Tuckwell

D.S.

, Farndale

R.W.

, and Barnes

M.J.

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens. J Biol Chem, 275, 35, 2000.

88.

Wojtowicz

A.M.

, Shekaran

, Oest

M.E.

, et al. Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair. Biomaterials, 31, 2574, 2010.

89.

Reyes

C.D.

, Petrie

T.A.

, Burns

K.L.

, Schwartz

, and García

A.J.

Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Biomaterials, 28, 3228, 2007.

90.

Clark

A.Y.

, Martin

K.E.

, García

J.R.

, et al. Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities. Nat Commun, 11, 114, 2020.

91.

Saito

, Suzuki

, Ogata

, Ohtsuki

, and Tanihara

Activation of osteo-progenitor cells by a novel synthetic peptide derived from the bone morphogenetic protein-2 knuckle epitope. Biochim Biophys Acta, 1651, 60, 2003.

92.

Goetinck

P.F.

, Stirpe

N.S.

, Tsonis

P.A.

, and Carlone

The tandemly repeated sequences of cartilage link protein contain the sites for interaction with hyaluronic acid. J Cell Biol, 105, 2403, 1987.

93.

Chow

L.W.

, Armgarth

, St-Pierre

J.-P.

, et al. Peptide-directed spatial organization of biomolecules in dynamic gradient scaffolds. Adv Healthc Mater, 3, 1381, 2014.

94.

Butterfield

K.C.

, Conovaloff

, Caplan

, and Panitch

Chondroitin sulfate-binding peptides block chondroitin 6-sulfate inhibition of cortical neurite growth. Neurosci Lett, 478, 82, 2010.

95.

Pillutla

R.C.

, Hsiao

, Beasley

J.R.

, et al. Peptides identify the critical hotspots involved in the biological activation of the insulin receptor. J Biol Chem, 277, 22590, 2002.

96.

Kopesky

P.W.

, Vanderploeg

E.J.

, Kisiday

J.D.

, Frisbie

D.D.

, Sandy

J.D.

, and Grodzinsky

A.J.

Controlled delivery of transforming growth factor β1 by self-assembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2/3 signaling. Tissue Eng Part A, 17, 83, 2011.

97.

Kopesky

P.W.

, Byun

, Vanderploeg

E.J.

, Kisiday

J.D.

, Frisbie

D.D.

, and Grodzinsky

A.J.

Sustained delivery of bioactive TGF-β1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells. J Biomed Mater Res A, 102, 1275, 2014.

98.

Kisiday

J.D.

, Colbath

A.C.

, and Tangtrongsup

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self-assembling peptide hydrogel. J Orthop Res, 37, 1368, 2019.

99.

Shao

, Zhang

, Pi

, et al. Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo. Biomaterials, 33, 3375, 2012.

100.

Speer

J.E.

, Barcellona

M.N.

, Lu

M.Y.

, et al. Development of a library of laminin-mimetic peptide hydrogels for control of nucleus pulposus cell behaviors. J Tissue Eng, 12, 1, 2021.

101.

Bridgen

D.T.

, Fearing

B.V.

, Jing

, et al. Regulation of human nucleus pulposus cells by peptide-coupled substrates. Acta Biomater, 55, 100, 2017.

102.

Barcellona

M.N.

, Speer

J.E.

, Jing

, et al. Bioactive in situ crosslinkable polymer-peptide hydrogel for cell delivery to the intervertebral disc in a rat model. Acta Biomater, 131, 117, 2021.

103.

OrthoTrophix

Inc

. A randomized, double-blind, placebo-controlled, study evaluating the safety and efficacy of a second course of intra-articular injections of TPX-100 in subjects who previously received

TPX-100 for patellar osteoarthritis involving both knees

. clinicaltrials. gov

September 2017

. Report

. NCT02837900. https://clinicaltrials.gov/ct2/show/NCT02837900 (accessed August 30, 2021).

104.

OrthoTrophix

Inc

. A randomized, double-blind, placebo-controlled, multi-dose phase 2 study evaluating the safety and efficacy of intra-articular injections of

TPX-100 in subjects with mild to moderate patello-femoral osteoarthritis involving both knees

. clinicaltrials. gov

April 2017

. Report

. NCT01925261. https://clinicaltrials.gov/ct2/show/NCT01925261 (accessed August 30, 2021).

105.

OrthoTrophix

Inc

. A prospective observational study to evaluate long-term changes in cartilage morphology in subjects who previously received TPX-100 or placebo in study

TPX-100-1 for patellar osteoarthritis involving both knees

. clinicaltrials. gov

April 2018

. Report

. NCT03125499. https://clinicaltrials.gov/ct2/show/NCT03125499 (accessed August 30, 2021).

106.

McGuire

, Lane

, Segal

, et al. TPX-100 leads to marked, sustained improvements in subjects with knee osteoarthritis: pre-clinical rationale and results of a controlled clinical trial. Osteoarthritis Cartilage, 26, S243, 2018.

107.

Engedi1000

E1K

, Ensol

Biosciences

, Inc. http://www.ensolbio.co.kr/eng/pipe_E1K.html (accessed August 30, 2021).

108.

Acar

, Srivastava

, Chung

E.J.

, et al. Self-assembling peptide-based building blocks in medical applications. Adv Drug Deliv Rev, 110, 65, 2017.