In Situ Bioprinting Embryonic-Derived Stem Cells to Repair Human Ex Vivo Chondral Defects

Abstract

Successful bioprinting requires an appropriate combination of bioinks, cells, and a delivery platform. To demonstrate the potential of in situ bioprinting for regeneration of cartilage lesions we combined clinically relevant embryonic-derived mesenchymal stem cells (ES-MSCs) with a fibrin-based bioink that was delivered into chondral defects created in human ex vivo osteoarthritic (OA) tissue using a bioprinting platform. We used an integrated multitool, 6-axis bioprinting system to laser scan and map the surface of chondral defects and bioprint within the cartilage defects in vitro and ex vivo. For cartilage neotissue generation, clinically relevant ES-MSCs were encapsulated at 20 × 10⁶ cells per mL in chondro-inductive bioinks composed of fibrinogen mixed with nanocellulose or fibrinogen mixed with nanocellulose and hyaluronic acid. After bioprinting as free-standing constructs or in situ within chondral defects, gels were cross-linked in thrombin and cultured for up to 8 weeks in chondrogenic medium. Print fidelity was assessed in the free-standing printed constructs after cross-linking and culture. In situ bioprinted constructs were evaluated for cell viability, mechanical properties, histology (Safranin O and collagen type II immunostaining), and gene expression of chondrogenic genes. Adding nanocellulose to fibrinogen significantly improved print fidelity. ES-MSCs in the fibrinogen-based bioink formulations generated cartilage-like neotissues with positive Safranin O and collagen type II staining. Chondrogenic genes (COLA2A1, ACAN, COMP, and SOX9) were significantly upregulated with negligible expression of hypertrophic markers (COL10A1 and RUNX2). The mechanical properties of the printed constructs increased from 30 to 50 kPa after 3 weeks to ∼150 kPa after 8 weeks in culture. We demonstrated the feasibility of combining clinically relevant ES-MSCs with printable fibrin-based hydrogel bioinks and an integrated bioprinting platform for in situ bioprinting that promoted neocartilage tissue generation and repair of ex vivo lesions in human OA tissues.

Impact Statement

A successful bioprinting approach for cartilage repair requires a combination of cells, bioinks, and a suitable platform. Here, we show the feasibility of combining clinically relevant ES-MSCs with printable fibrin-based hydrogel bioinks and an integrated bioprinting platform for in situ bioprinting that promoted neocartilage tissue generation and repair of ex vivo lesions in human osteoarthritic tissues. Our integrated approach represents the basis to advance bioprinting for cartilage and osteochondral regeneration and holds promise to deliver the appropriate cells and bioinks for tissue regeneration directly in vivo.

Keywords

stem cells bioprinting chondral defects

Introduction

Bioprinting is an advanced tissue fabrication technique that involves deposition of biological materials, including living cells, to create complex three-dimensional (3D) structures that can mimic natural tissues. Bioprinting offers precision and customization which has potential for medical applications such as tissue repair, drug testing, and regenerative medicine. Successful bioprinting requires an optimal combination of variables to lead to the formation of the target neotissue. The three major challenges are identifying an appropriate cell source, formulating a suitable bioink to deliver the cells, and developing a compatible bioprinting platform. The ideal cell source is one that is readily available in large numbers that can survive the 3D printing process and is able to differentiate into the desired stable phenotype for long-term neotissue survival, especially in vivo. Bioinks need to be biocompatible and printable, generate the appropriate material properties after deposition, and provide the biochemical and biophysical cues to support or even enhance the desired cellular activity and tissue formation. Lastly, the 3D printing system must be compatible with the bioink, the cells, and support the feasibility of delivering the cellular bioinks to its intended location with the required spatial accuracy.

A range of cell sources have been investigated for engineering cartilage. While chondrocytes are attractive and have been extensively researched, they are available only in limited quantities and often lose the capacity to form quality neotissue after culture expansion.^1–3 Progenitor cells harvested from articular cartilage have been used in 3D bioprinting as an alternative cell source.^4,5 However, harvesting these cells for clinical translation is challenging. Other progenitor cells such as bone marrow-derived mesenchymal stem cells (MSCs),⁶ adipose-derived MSCs,^7,8 human umbilical cord blood-derived MSCs,⁹ embryonic stem cell-derived MSCs (ES-MSCs),¹⁰ and induced pluripotent stem cells (iPSCs)¹¹ have also been utilized in cartilage bioprinting studies. Each of these sources have pros and cons relevant to tissue harvesting morbidity and restrictions, cartilage forming capacity, and ethical and safety concerns.

The choice of bioink is essential for the success of 3D printing of microenvironments to promote the formation of cartilaginous tissues. Hydrogels are ideal for bioprinting because their water content mimics the natural extracellular matrix, their mechanical properties can be tailored for precise control over stiffness, and they are easily modified to include bioactive molecules.¹² Several hydrogels possess the biological features necessary for enhancing cell adhesion, proliferation, and differentiation to create an extracellular microenvironment to support key functions such as cell attachment, proliferation, and differentiation.¹³ To balance constraints on viscosity imposed by bioprinting systems with shape fidelity and mechanical properties after printing, rapid hydrogel gelling or cross-linking methods are required to have minimal impact on cell viability. These methods include photocross-linking,^14,15 temperature change,¹⁶ chemical cross-linking,¹⁷ ionic cross-linking,¹⁸ and enzymatic cross-linking.¹⁹

As no single hydrogel has all the desirable qualities necessary for successful bioprinting of cartilage, biomaterials with complementary attributes have been combined to address specific deficiencies. Adding gellan gum to methacrylated gelatin (GelMA) significantly increased the viscosity and speed of gelation²⁰ and improved mechanical properties after printing.²¹ Nanofibrillated cellulose or nanocellulose (NC) when combined with alginate improved printability and supported cartilage-like tissue formation.^11,22–26 Combining NC with GelMA and methacrylated hyaluronic acid induced cartilage-specific gene expression.²⁷

Fibrin is a natural polymer approved by the U.S. Food and Drug Administration (FDA) for broad clinical applications²⁸ and can support cartilage regeneration.^29–31 Because fibrinogen is readily cross-linked by thrombin to form fibrin, there have been several efforts to improve printability and postprint stability. To support cartilage formation, fibrinogen has been combined with collagen,³² alginate,³³ cartilage extracellular matrix (ECM),^34,35and hyaluronic acid.^36,37 In our study. we explored combining NC and hyaluronic acid (Hy) with fibrin.

“In situ” bioprinting involves printing directly into the site of injury or disease and has several advantages including patient-specific or lesion-specific customization, real-time adjustment during surgery, and potential for better tissue integration.^1,38–40 In situ bioprinting requires sophisticated integration of robotics, bioink deposition, and 3D scanning.⁴¹ The BioAssemblyBot (BAB) (Advanced Solutions, Louisville, KY) is a 6-axis bioprinting platform combining pneumatic-driven extrusion-printing, laser scanning, and temperature control that supports multiple print tools with automated tool changes. This system has been used to demonstrate potential for applications ranging from burn healing to gel stiffness tuning.^42,43

In this study, we bioprinted ES-MSCs encapsulated in custom bioinks into chondral defects created in osteoarthritic cartilage tissue using an integrated 6-axis bioprinting platform. We demonstrated the following proof of concept for in situ bioprinting: (i) biocompatibility of a clinically relevant cell source encapsulated at high cell density in a printable hydrogel, (ii) delivery into ex vivo human tissue defects using a multiaxial bioprinting platform, and (iii) formation of a robust neocartilage tissue after ex vivo culture.

Method

Six-axis pneumatic 3D printer and integrated laser scanner

We used a multitool, extrusion-based system capable of manipulating each tool with 6 degrees of freedom (BAB, Fig. 1). A pneumatic extrusion tool was used to deposit bioink onto the print substrate. We optimized the nozzle diameter, pneumatic pressure, and tool speed to achieve the most consistent bioink extrusion.

FIG. 1.

Overview of printing system with specific tools required for scanning and bioink extrusion. (A) The BioAssemblyBot (BAB) printing system with a 6-axis robotic arm and an assortment of tools for printing. (B) The laser scanner tool was used for scanning cartilage defects. (C) A pneumatic extrusion printing tool was used for delivery of fibrin-based hydrogel-based bioink.

A 3D surface map of the articular cartilage surface was captured with a laser scanner (15 µm resolution) mounted on the printer arm to register the scanned features within the print space. The surface of the articular cartilage defect was modeled using this surface map in the TSIM software (Advanced Solutions, Louisville, Fig. 2). A tool path for printing was generated using custom in-house software with path spacing calculated based on the extrusion diameter of the bioink.

FIG. 2.

Fibrin gel printability and delivery into a cartilage lesion. (A) 5 mm cubes were printed to check shape fidelity (N = 4). Average corner radius of 1.16 mm. (B–C) Monitoring retention of printed shape fidelity with bioink containing embryonic-derived mesenchymal stem cells (ES-MSCs) over 21 days of chondrogenic culture. (D–F) Illustration of robotic arm with laser scanner tool used to map the topography of a chondral lesion. (E) After laser scanning the tissue lesion, the scanned model is used to generate the print path to fill the specific three-dimensional geometry of the lesion. (F) Illustration of robotic arm with the pneumatic print head with bioink with cells being deposited into the cartilage lesion. (G–H) Laser scanning of an irregular lesion created in an osteoarthritic (OA) osteochondral explant harvested from a patient undergoing total knee arthroplasty. The shape and depth of the lesion was reconstructed, and the defect was repaired (I–J) using the robotic arm and syringe tool containing a fibrin-based hydrogel. The blue outlined regions denoted the lesion before (H) and after (J) printing.

Cell and tissue sources

Human embryonic stem cells (ESC) were differentiated into mesenchymal stem cells (ES-MSCs) as previously described.⁴⁴ Briefly, xeno-free derived ESC (HADC-100 ESC⁴⁵) were suspended in spinner culture flasks to form small cell clusters. The cell clusters were cultured for 5 days in the presences of an ALK-5 inhibitor (10 µM; SB525334; Selleckchem, Houston, TX) and then seeded for adherence onto fibronectin-coated flasks in serum free medium (StemPro34, Thermo Fisher Scientific, Carlsbad, CA) supplemented with basic fibroblast growth factor (bFGF) (20 ng/mL). Cells that emerged from the seeded clusters were passaged several times and cryopreserved in liquid nitrogen. These cells expressed MSC surface markers (CD73, CD90, and CD105 positive; CD34 and CD45 negative) and were highly chondrogenic (pellet cultures and ex vivo defect repair⁴⁴). ES-MSCs were used in this study between passages 3 and 5.

Osteochondral tissues were obtained from patients undergoing total knee arthroplasty (TKA) (n = 7; 4 females and 3 males; average age 69 ± 7 years) within 4–6 hrs of surgery (approved by Institutional Review Board, Scripps Health, CA). Osteochondral tissues were prepared for either direct bioprinting (with intact bone and cartilage) to optimize delivery of bioinks into osteochondral lesions or for chondral bioprinting. For chondral only experiments, articular cartilage (without bone) was harvested for explant culture to document the performance of the bioinks in surgically created chondral lesions. Before bioprinting, the cartilage explants were aseptically cultured in culture medium consisting of Dulbecco’s modified Eagle medium (DMEM) (Mediatech Inc., Manassas, VA) supplemented with 10% calf serum (Omega Scientific Inc., Tarzana, CA) and 1% penicillin–streptomycin–gentamycin (Life Technologies, Carlsbad, CA).

Bioink formulation and cell density

We tested two bioink formulations. The first formulation, fibrinogen mixed with nanocellulose (FiNC) consisted of cells (20 × 10⁶/mL), suspended in serum-free chondrogenic medium supplemented with transforming growth factor beta 3 (TGFβ3) (10 ng/mL, Peprotech, Rocky Hill, NJ), and mixed with bovine plasma fibrinogen (Type I-S, 65–85% protein, Sigma-Aldrich Corp. St. Louis, MO) at a final concentration of 60 mg/mL and 1.9% (w/v) nanofibrillated cellulose (NC, CELLINK LLC, Boston, MA). The serum-free chondrogenic medium consisted of DMEM, 1× ITS + 1 supplement (Sigma-Aldrich), 100 µM ascorbic acid 2-phosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), 1.25 mg/mL human serum albumin (Bayer, Leverkusen, Germany), and 1% penicillin–streptomycin–gentamycin. For the second formulation, fibrinogen was mixed with nanocellulose and hyaluronic acid (FiNC+Hy); 1mg/mL hyaluronic acid (Supartz, Seikagaku Corporation, Tokyo, Japan) was added to the chondrogenic medium used to suspend the cells and fibrinogen, maintaining the same final cell density as for the FiNC bioink.

Printing fidelity

To determine the print fidelity of the fibrin and NC hydrogels, a series of 5 × 5 × 5 mm cubes were printed in a 6-well plate. Following cross-linking in 100 IU/mL thrombin (from bovine plasma, Sigma-Aldrich) for 20–30 min, the cubes were digitally imaged, and the corner radius of each printed cube was measured. We measured print fidelity for concentrations of fibrinogen ranging from 1.5% to 9% and selected a final concentration of 6% as optimal for handling and printability.

Bioprinting in situ

We used a dermal punch to manually create irregular chondral defects in ex vivo human cartilage explants, ∼2 mm in depth and 5–8 mm across. Each individual ex vivo tissue sample was placed on a sterile petri dish and the BAB laser scanning tool was used to scan the defect using a step size of 250 µm (Fig. 2). The scanned points were used to construct a 3D surface map of the defect, which was used to generate the print tooling path that followed the contours of the defect (Fig. 2E).

The FiNC gel with cells was loaded into an extrusion syringe barrel (Nordson, Westlake, OH) fitted with a 25 gauge luer lock needle. Cell-laden bioink was printed into the scanned ex vivo chondral defect (Fig. 2) and cross-linked in thrombin (100 IU/mL) for 20–30 min. The cross-linked constructs were cultured in serum-free chondrogenic medium supplemented with TGFβ3 (10 ng/mL) with medium changes every 3–4 days, for 3 or 8 weeks.

In parallel experiments, FiNC or FiNC+Hy were printed onto sterile petri dishes or 6-well plates in the shape of 5 mm free-standing cubes. These prints were used to assess the printing properties (i.e., fidelity, shape) as well as assessing the effect of culturing the different formulations for comparison with the ex vivo printed constructs. All printed constructs were cross-linked with thrombin and cultured for 3 weeks. Experiments were replicated by printing four different cell preparations in TKA cartilage tissues obtained from multiple donors (N = 6).

Mechanical testing

The mechanical properties of printed constructs and surrounding cartilage were measured using indentation testing 30 min after thrombin cross-linking and after 3 or 8 weeks of culture.⁴⁶ Specimens were indented to a depth of 150 µm with a round indenter with a diameter of 1.0 mm at a rate of 11.5 mm/min. An elastic modulus was calculated as described by Hayes et al.^47,48

Cell viability

Cell viability of printed constructs was assessed on at least three different experiments conducted for each condition after 3 weeks of culture. We used Calcein-AM and ethidium homodimer-1 (Live/Dead kit, Life Technologies, Carlsbad, CA) to stain the tissues, imaged on a laser confocal microscope (LSM-510, Zeiss, Jena, Germany) as previously described.⁴⁹ The percentage of live and dead cells was calculated using ImageJ/Fiji⁵⁰ after image processing by thresholding and segmentation as described previously.⁴⁶

Histology

All constructs were fixed in Z-Fix (ANATECH, Battle Creek, MI) for at least 48 h before dehydration and embedding into paraffin. Sections of 4 µm thickness were stained with Safranin-O and Fast Green to detect deposition of glycosaminoglycan (GAG) distribution in the printed tissues. For detection of collagen type II, all sections were pretreated with pepsin and 2 µg/mL mouse-anti human collagen type II (II-II6B3, Developmental Studies Hybridoma Bank, University of Iowa). Primary antibody-treated sections were placed into a humid box and incubated at 4°C overnight. The ImmPRESS secondary DAB kit (Vector Laboratories, Burlingame, CA) was used for color development. Human cartilage tissue was used as a positive control for collagen type II. Species-matched isotype controls were used to assess nonspecific staining.

Quantitative PCR

RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA) from monolayer cultured cells (to establish baseline gene expression), from free-cultured FiNC and FiNC+Hy constructs, and from some ex vivo printed FiNC and FiNC+Hy gels (after removing the gels from the defect before preparing for histology). Using total RNA as a template, cDNA was synthesized according to the manufacturer’s protocols (Applied Biosystems, Foster City, CA). Prevalidated TaqMan® gene expression reagents (Applied Biosystems) were used for qPCR to examine the expression of chondrogenic marker genes (COL1A1, COL2A1, COMP, and ACAN) and hypertrophic marker genes (COL10A1 and RUNX2). GAPDH was used to normalize gene expression levels, the ΔCt method was employed as previously reported,⁵¹ and the relative change in expression in each bioink and treatment were compared with the monolayer cultured cell expression levels.

Image analysis

Safranin O stained repair regions were quantified in free-cultured gels after 3 weeks of culture or in ex vivo cultures after 3 and 8 weeks in culture using ImageJ as detailed before.⁴⁴ Briefly, the red color channel was used to identify the glycosaminoglycans stained red with safranin O stain. A region of interest (ROI) was defined by manually outlining the printed gel within the ex vivo defect. After thresholding the red channel signal to remove background, the percent positive signal was recorded from the selected ROI.

Statistics

Comparisons between the mechanical properties, gene expression, and viability of FiNC and FiNC+Hy gels were made using a t-test, where a p value of <0.05 was considered significant. Difference in gene expression between ES-MSCs cultured in FiNC gels and cells in monolayer cultures was tested for significance at p < 0.05, using the online BootstRatio application (http://regstattools.net/br).⁵²

Experiment

Integration of BAB scanning and delivery of fibrin-based hydrogels in chondral lesions

The FiNC hydrogels were printed into 3D cubes of 5 × 5 × 5 mm and the radius of the corners of these printed blocks were measured to assess printing fidelity. We noted an average corner radius of 1.16 mm (N = 4) as shown in Figure 2A. These printed hydrogels cubes retained excellent morphology when combined with ES-MSCs (20 × 10⁶ cells/mL) over 21 days of culture (Fig. 2B–C).

The sequential steps of laser scanning to final print are outlined in Figure 2D to 2F. To demonstrate the utility of combining the laser scanner, software, and delivery of our bioink, we selected human osteoarthritic osteochondral tissue specimens with irregular chondral defects (Fig. 2G and H). These tissue specimens were scanned, modeled, and printed with bioink to fill the defect and to match the height of the surrounding tissue (Fig. 2I and J). The total time from the start of the scan to completion of the defect fill was ∼15 mins.

Cartilage-like phenotype supported in fibrin-based hydrogels

ES-MSCs were suspended into FiNC or FiNC+Hy gels at a cell density of 20 × 10⁶ cells per mL and bioprinted onto tissue culture plastic. Bioprinted constructs were cross-linked with thrombin for 20–30 min and cultured for 3 weeks in chondrogenic differentiation medium. Cell viability for both bioinks averaged 72 ± 8% (Fig. 3), with no significant differences between FiNC+Hy gels (76 ± 9%) or FiNC gels (70 ± 8%). After 3 weeks in culture, glycosaminoglycans (GAGs) were deposited principally around the cells, as revealed by red safranin O staining (Fig. 3C–H), and appeared more evenly distributed throughout the constructs in the FiNC+Hy gels (Fig. 3F–H). However, image analysis of the free-cultured FiNC and FiNC+Hy stained with Safranin O indicates no significant difference between these conditions (p = 0.1). The enhanced GAG deposition was reflected in the FiNC+Hy gene expression profiles (Fig. 3I) with higher ACAN and COMP expression (although not significantly different from the FiNC condition (p < 0.1). High expression of COL2A1 in both FiNC and FiNC+Hy conditions are indicative of a chondrogenic phenotype. COL10A1 expression (a marker of hypertrophic differentiation) was significantly higher in the FiNC+Hy gels (p < 0.01; Fig. 3I), while the transcription factor, RUNX2 (another hypertrophic marker), was ∼2-fold lower relative to the monolayer cultured ES-MSCs (baseline) in both conditions, although not statistically significant (p < 0.1; Fig. 3J).

FIG. 3.

Bioprinted embryonic-derived mesenchymal stem cells (ES-MSCs) in fibrinogen mixed with nanocellulose (FiNC) and fibrinogen mixed with nanocellulose and hyaluronic acid (FiNC+Hy) gels show viable neocartilage formation with glycosaminoglycan deposition and chondrogenic gene expression after 3 weeks in culture. Confocal live/dead image of ES-MSCs in (A) FiNC gel or (B) FiNC+Hy gel. Safranin O staining of ES-MSCs in FiNC gels (C–E) and FiNC+Hy gels (F–H). (I–J) Quantitative qPCR of chondrogenic and osteogenic gene expression in FiNC (N = 14) and FiNC+Hy (N = 10) gels. (*p < 0.01).

Evidence of chondral defect repair

ES-MSCs suspended in FiNC or FiNC+Hy gels were bioprinted into ex vivo OA human cartilage tissues (Fig. 4A–B). After 3 weeks in chondrogenic medium, a stable and integrated tissue was formed in both FiNC (Fig. 4A) and FiNC+Hy (Fig. 4B) gels. No significant difference in mechanical properties was observed between the FiNC and FiNC+Hy conditions (elastic modulus averaging 44.2 ± 11.8 Kpa for FiNC and 43.2 ± 18.3 Kpa FiNC+Hy gels, Fig. 4C). Safranin O staining showed deposition of cartilage-like matrix in both hydrogel formulations (Fig. 5), especially close to the interface between the host cartilage tissue (Fig. 5A and B), which was reflected in the higher deposition of collagen type II immunostaining intensity (Fig. 5). For gene expression analysis, a portion of hydrogel was carefully removed from the bioprinted TKA defects after 3 weeks in culture (Fig. 4A and B). The expression profile of chondrogenic genes (Fig. 5C and D) in ex vivo printed tissues was similar to that in free-cultured tissues (Fig. 3I and J). We found significantly lower COMP (p < 0.001), COL1A1 (p < 0.01), and RUNX2 (p < 0.01) expression in the FiNC gels relative to the FiNC+Hy gels (Fig. 5C and D).

FIG. 4.

Macro images and mechanical properties of cultured human OA cartilage explants with (A) fibrinogen mixed with nanocellulose (FiNC) and (B) fibrinogen mixed with nanocellulose and hyaluronic acid (FiNC+Hy) hydrogels with embryonic-derived mesenchymal stem cells (ES-MSCs). Sections were processed separately for histology and for gene expression. (C) Mechanical properties after 3 weeks in culture: FiNC = 44 ± 12 kPa (N = 6) and FiNC+Hy = 43 ± 18 kPa (N = 9); FiNC after 8 weeks of culture (123 ± 68 kPa; N = 4), in comparison with surrounding the native osteoarthritic tissue (444 ± 308 kPa, N = 4). (*p < 0.04; **p < 0.03).

FIG. 5.

Histology, immunostaining, and gene expression profiling of 3-week cultured human OA cartilage explants bioprinted with either fibrinogen mixed with nanocellulose (FiNC) or fibrinogen mixed with nanocellulose and hyaluronic acid (FiNC+Hy) hydrogels with embryonic-derived mesenchymal stem cells (ES-MSCs). (A) FiNC Safranin O and collagen type II immunostaining of explants and hydrogels with ES-MSCs. (B) FiNC+Hy Safranin O and collagen type II immunostaining of explants and hydrogels with ES-MSCs. (C–D) Gene expression profiles of neotissues extracted from bioprinted TKA defects. (N = 9 FiNC and N = 6 FiNC+Hy).

As the FiNC+Hy formulation did not significantly improve neotissue formation, we analyzed a longer term culture with the FiNC formulation. We printed ES-MSCs in FiNC gels into ex vivo chondral defects (N = 4 explants) and cultured the constructs for 8 weeks. The neocartilage tissues formed displayed more intense Safranin O staining (Fig. 6A–C) and greater collagen type II deposition throughout the construct (Fig. 6D–E) compared with the 3 week ex vivo cultures. At 8 weeks this tissue also exhibited significantly higher mechanical properties (122.8 ± 67.6 kPa, p < 0.005) in comparison with the 3-week FiNC (44.2 ± 11.8 Kpa) or FiNC+Hy (43.2 ± 18.3 Kpa) tissues (Fig. 4C). Image analysis of FiNC hydrogel printed ex vivo indicate a greater Safranin O staining intensity (GAG deposition) after 8 weeks of culture (87.0 ± 13.5%) compared with 3 week cultures (11.0 ± 1.9%).

FIG. 6.

Histology of 8-week cultured embryonic-derived mesenchymal stem cells (ES-MSCs) in fibrinogen mixed with nanocellulose (FiNC) bioinks printed into defects in ex vivo human OA cartilage. Transverse sections of the defects displayed complete filling of the irregular defects in the host tissue (HT) with (A–C) the printed tissue (PT) showing deposition of glycosaminoglycans as observed with Safranin O positive staining and with (D–E) ECM rich in collagen type II. (F) Isotype control for collagen type II immunostaining.

Discussion

For successful bioprinting, an appropriate combination of cells, bioink materials, and a suitable bioprinting platform is essential. Here we demonstrated the feasibility of combining stem cells in a suitable bioink in a delivery platform to explore the potential of in situ bioprinting as a valid approach to repair cartilage lesions. We delivered clinically relevant ES-MSCs using custom printable fibrin-based hydrogels into ex vivo chondral defects using a platform that integrated laser scanning of articular defects with a 6-axis bioprinting arm. The bioprinted hydrogels showed potential for neocartilage tissue generation and repair of ex vivo lesions in human osteoarthritic tissues.

A variety of MSCs sources have been used as promising sources of chondroprogenitors for cartilage regeneration including MSCs harvested from adipose tissue,^53,54 bone marrow,⁵⁵ and umbilical cord.^56,57 However, limitations in cell proliferation, donor-to-donor variation, phenotype stability, senescence, and the tendency to undergo hypertrophy remains an issue.^58–60 ES-MSCs represent an attractive cell source for cartilage regeneration because of very high capacity for proliferation and immunomodulatory properties. Our results of reduced COL10A1 and RUNX2 expression are consistent with the reduced risk of hypertrophic differentiation in ES-MSCs.^44,61 The ES-MSCs used in this study are clinically relevant (e.g., derived under xeno-free conditions), tolerated the bioink formulation and the bioprinting process, and generated neocartilaginous tissue with promising qualities.

Our goal was to develop a bioink formulation from components that are in clinical use and can be printed with clinically safe cross-linking methods (thrombin). While several bioink formulations been reported for extrusion bioprinting, many of these require modifications for cross-linking after printing, for example, methacrylation of gelatin^20,21,62 or hyaluronic acid^27,40,63 and require the addition of photocross-linkers and exposure to UV light with the concomitant risk of phototoxicity. Fibrinogen by itself is not suitable for extrusion bioprinting. Several agents, such as gelatin, polyethylene glycol, and alginate have been used for enhancing the printability of fibrinogen.^64–66 We selected NC to enhance the printability of fibrinogen, due to its shear thinning behavior as a hydrogel and relative safety for clinical applications.^67,68

NC is a widely abundant and relatively low-cost material that supports cartilage tissue formation.⁶⁹ NC is considered nontoxic, nonimmunogenic, noninflammatory, and enables cells to readily adhere, proliferate, and differentiate.^70–72 The U.S. FDA generally recognizes NC and other nano-sized cellulose as safe.⁶⁸ Several studies have explored the potential of NC for treating skin injuries⁷³ and bone tissue engineering.^74,75 NC-based treatments are also in clinical trials and listed on clinicaltrials.gov.⁶⁸ Collectively, these research studies, commercial use, and clinical trials support the safety and potential efficacy of NC.

Adding NC to alginate enhances print fidelity, shape retention, and compressive stiffness after ionic (calcium chloride) cross-linking.²² Chondrogenic tissue formation has been reported after bioprinting iPSC and irradiated chondrocytes,¹¹ or nasal chondrocytes, alone or in combination with bone marrow-derived MSCs,²⁵ in NC mixed with alginate hydrogel. We observed cell viability of ∼70% after 3 weeks in culture, similar to that reported by others.²² The compressive stiffness of our cultured bioprinted FiNC constructs increased from 50 kPa after 3 weeks to 150 kPa after 8 weeks. Our data demonstrates NC to be a favorable additive for extrusion printing and for supporting neocartilage-like tissue formation.

The major advantages of bioprinting in situ include the feasibility of complex tissue fabrication, patient-specific or lesion-specific customization, real-time adjustment during surgery, and potential for better tissue integration. Feasibility of in situ printing with acellular inks has been shown in animal cadaver tissues (alginate and demineralized bone matrix),³⁸ alginate, and methacrylated polyethylene glycol and hyaluronic acid in rabbit and mini-pig.³⁹ In situ bioprinting with cellular bioinks been reported in vivo in cranial defects in rats⁷⁶ and trochlear defects in rabbit femurs.⁴⁰ We had previously reported on the results of in situ bioprinting human chondrocytes suspended in methacrylated polyethylene glycol in ex vivo bovine osteochondral defects using ink-jet printing.¹ In this study, we developed a clinically relevant bioink using an integrated platform capability of scanning and printing directly into cartilage lesions and documented proof of concept of in situ chondrogenic repair. However, all these potential advantages must be rigorously tested in preclinical in vivo studies and validated in clinical trials.

Our study has the following weaknesses. We did not show that bioprinting was better than manually injecting the hydrogel into the defect. NC is biocompatible; however, the long-term fate of the NC particles and any potential deleterious effect of NC particles released within the knee joint needs to be considered. Although we printed into osteochondral explants, we only attempted repair of cartilage defects. Repairing osteochondral defects will also require developing and testing bioinks for repairing bone. Finally, all in vitro and ex vivo results must be validated in vivo.

Conclusions and Future Directions

In this study, we demostrated the feasibility of combining clinically relevant ES-MSCs with printable fibrin-based hydrogel bioinks and an integrated bioprinting platform for in situ bioprinting that promoted neocartilage tissue generation and repair of ex vivo lesions in human osteoarthritic tissues.

Further work is needed to print layers emulating the layers of articular cartilage. Reconstructing the subchondral bone will require osteogenic cells and bioinks. Finally, convincing evidence is needed to compare the safety, efficacy, and cost of in situ bioprinting to clinically available alternatives such as implantation of prefabricated grafts for cartilage repair, autografts, or allografts.

Authors’ Contributions

S.P.G., E.W.D., and D.D.D. were responsible for the overall experimental design. S.P.G. and D.D.D. wrote the article in close collaboration with the other authors. N.E.G. maintained and cultured the cells and 3D cultures, performed ex vivo studies, viability assessments, and coordinated histological studies. E.W.D. performed all scanning, modeling, bioprinting, mechanical testing, and histomorphometry. S.P.G. coordinated and performed qPCR characterizations. All authors discussed the results and approved the final version of the article.

Footnotes

Acknowledgments

The authors thank April Damon and Jessica Edwards for technical assistance with gene expression, histology, and immunostaining. The authors are grateful for the editing by Emily Martin. The authors greatly appreciate the support of the Shaffer Family Foundation and Donald and Darlene Shiley.

Funding Information

This work was funded by CIRM (PC1-08128), the Shaffer Family Foundation, and Donald and Darlene Shiley.

Disclosure Statement

No competing financial interests exist.

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