State of the Art of Clinical Applications of Tissue Engineering in 2021

Abstract

Tissue engineering (TE) was introduced almost 30 years ago as a potential technique for regenerating human tissues. However, despite promising laboratory findings, the complexity of the human body, scientific hurdles, and lack of persistent long-term funding still hamper its translation toward clinical applications. In this report, we compile an inventory of clinically applied TE medical products relevant to surgery. A review of the literature, including articles published within the period from 1991 to 2020, was performed according to the PRISMA protocol, using databanks PubMed, Cochrane Library, Web of Science, and Clinicaltrials.gov. We identified 1039 full-length articles as eligible; owing to the scarcity of clinical, randomized, controlled trials and case studies, we extended our search toward a broad surgical spectrum. Forty articles involved clinical TE studies. Among these, seven were related to TE protocols for cartilage applied in the reconstruction of nose, ear, and trachea. Nine articles reported TE protocols for articular cartilage, nine for urological purposes, seven described TE strategies for cardiovascular aims, and eight for dermal applications. However, only two clinical studies reported on three-dimensional (3D) and functional long-lasting TE constructs. The concept of generating 3D TE constructs and organs based on autologous molecules and cells is intriguing and promising. The first translational tissue-engineered products and techniques have been clinically implemented. However, despite the 30 years of research and development in this field, TE is still in its clinical infancy. Multiple experimental, ethical, budgetary, and regulatory difficulties hinder its rapid translation. Nevertheless, the first clinical applications show great promise and indicate that the translation toward clinical medical implementation has finally started.

Impact statement

The clinical use of a tissue-engineered windpipe in compassionate patients elicited euphoria in the media between 2010 and 2016: tissue engineering (TE) had proven to be no longer a fictional concept but a life-saving reality. However, most of the treated patients died, and the surgeon was convicted for scientific misconduct and aggravated assault. As of 2020, the authors had eight of their articles retracted and two received an expression of concern.¹ These events have fueled skepticism among clinicians about TE: science or fiction?

Although TE is full of promises, it is not realistic yet to engineer a (vascularized) construct that thrives in a “hostile” clinical environment. Therefore, a realistic update of the current clinical outcomes and the promises of TE in human trials are essential.

Introduction

Langer and Vacanti define tissue engineering (TE) as “an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.”² As proof of principle, Vacanti et al. grafted a human auricle-shaped polymer-cell construct onto the back of a mouse in 1991.³ They introduced TE into animal experiments using chondrocytes from newborn calf joints seeded on a synthetic scaffold for culture.⁴ After these were cultured, the construct with cultured cells was implanted subcutaneously in nude mice. Their work demonstrated the fascinating possibility of creating new autologous cartilage on a synthetic scaffold in an animal model and marked the onset of the field of “TE.”

Subsequently, considerable efforts have been expended to implement TE in clinical practice. TE refers to methods used to synthesize tissue substitutes created with a matrix populated by autologous cells and their subsequent implantation in vivo. To provide this matrix, one can synthesize allogeneic tissue and perform an extensive decellularization process that results in an acellular matrix, which is then recellularized with autologous cells, commonly referred to as the “decell–recell” concept.⁵ Alternatively, a matrix can be fabricated with synthetic or biological materials, which can then be seeded with autologous cells.

As documented by the number of hits that appear on the term “TE” in PubMed, interest has grown tremendously since the 1990s.⁶ However, three decades since the first report of Vacanti et al.,³ clinical studies are very scarce and do not yet meet the expectations set 30 years ago. For tissues that do not need intrinsic vascularization, such as bone and articular cartilage, stability against clinical forces—traction, friction, and pressure—is mandatory, whereas for elastic cartilage flexibility and shape are essential. For tissues that require revascularization, the regeneration of neovascular networks within a three-dimensional (3D) matrix to distribute oxygen and other nutrients within the TE device is the major hurdle. It is very challenging to rebuild the capillary network within the TE organ. Preclinical models have shown that implanted TE construct get nutrients originally from tissue fluid permeation and secondarily from neovascularization originated from enclosed tissue. The center area of the TE construct experiences ischemia at the beginning of transplantation. Therefore, it is possible that a main part of the seeded cells would not survive and participates in degradation of the construct. In a clinical surgical setting, we can alter vascularization by prefabrication and prelamination strategies, which may lead to hybrid “ex vivo” approaches using principles of cell engineering and growth factor therapy,^7–9 decellularization of scaffolds, and microvascular surgery.^10–12 If intrinsically vascularized tissues do not rapidly receive a source of vascularization tissue necrosis occurs in a clinical setting.^13,14

This review focuses on all clinical studies currently conducted with tissue engineered medical products in a clinical setting.

Methods

Data search protocol

In accordance with the PRISMA guidelines, a systematic search was performed using PubMed, Cochrane Library, Web of Science, and Clinicaltrials.gov to identify pilot clinical studies and reports from compassionate-use programs published since the first trial conducted by Vacanti on August 1, 1991 until the end date of the search in August 2020.

The Patient, Intervention, Comparison, Outcome (PICO) question was stated as: ((“Clinical Trial” [Publication Type] OR clinical-trial[tiab] OR intervention-study[tiab] OR “Clinical Study” [Publication Type] OR Clinical-study[tiab] OR “Clinical Trial, Veterinary” [Publication Type] OR ((“Patients”[Mesh] OR Patient[tiab] OR large-animal*[tiab] OR “Sheep”[Mesh] OR sheep[tiab] OR ovis[tiab] OR “Goats”[Mesh] OR goat*[tiab] OR “Swine”[Mesh] OR swine[tiab] OR porcine[tiab] OR pig[tiab] OR pigs[tiab] OR “Cattle”[Mesh] OR cattle[tiab] OR cow[tiab] OR bovine-animal[tiab] OR “Horses”[Mesh] OR horse*[tiab] OR equine-animal[tiab] OR “Dogs”[Mesh] OR dog[tiab] OR dogs[tiab] OR “Rabbits”[Mesh] OR rabbit*[tiab]) AND in-vivo[tiab] OR implant*[tiab]))) AND (“Tissue Engineering”[Mesh] OR tissue-engineer* [tiab] OR recell*[tiab]).

Endnote X9^® (Clarivate Analytics, Philadelphia, PA) was used as reference manager. The selection of articles was conducted by two independent reviewers. Reference lists of included articles were hand-searched for possibly suitable articles. Inter-examiner conflicts were dissolved by discussion until a definitive list of eligible articles was composed.

Eligibility criteria

Studies meeting the following predefined criteria were included: (1) clinical trials in humans including randomized clinical trials, cohort studies, case–control studies, cross-sectional studies, and case series; (2) full text available in the English language; (3) TE¹⁵ (macroscopic scaffold seeded with autologous cultured cells) was the main subject; (4) study subjects were patients treated with TE constructs; (5) studies published within the period from August 1, 1991 to August 1, 2020. Following criteria were used for exclusion from current review: (1) full-text not available; (2) non-English text; (3) in vitro studies; (4) systematic reviews, meta-analyses, and commentaries; and (5) studies on acellular grafts and with cellular implants without any graft or scaffold.

Data collection

Data extraction of included articles followed. These were analyzed and reported using MS Office Excel 2016. The data recorded were first author, year, study type, number of included patients, treatment technique, treatment materials, follow-up time, and outcomes. No statistical analysis was conducted, owing to the broad array of parameters.

Results

The complete full-search strategy is schematically summarized in Figure 1.

FIG. 1.

PRISMA flow diagram. PRISMA flow diagram used for scoping review. *Additional sources found by hand searching key journals for the topic of tissue engineering and searching table of contents, as well as reviewing reference lists form our selected articles and citing articles. Color images are available online.

This review referenced a total of 40 studies with different study characteristics and designs. Clinical studies in which TE was used for plastic reconstructive purposes were considered the most relevant (seven studies; Table 1). These studies covered the reconstruction of nose, ear, and tracheal cartilage with TE substitutes. Nine studies reporting articular cartilage TE were subjected to full-text screening and were reviewed in this survey (Table 2). Nine clinical studies reported the treatment of urological defects with TE (Table 3), seven clinical studies described cardiovascular TE (Table 4), and eight clinical studies focused on treatment of mucosal defects (Table 5).

Table 1.

Status of Clinical Translation of Tissue Engineering in Plastic Reconstructive Surgery

First author, Year	Cell source	Scaffold	Time in vitro^*	Time in vivo	Model	Observations
Elliott et al. (2012)³⁰	Autologous BM-MNCs	Decellularized homograft	None	4 Y FU	Compassionate pediatric patient	Trachea crisis protocol: intraoperative cell seeding hrEPO + omental flap to support angiogenesis TGF-β1 + G-CSF for chondrocyte differentiation and autologous MSC recruitment Stenting for 2Y Maturation of epithelium → decrease in the frequency of dilatation therapies
Fulco et al. (2014)⁴⁰	Autologous chondrocytes from the nasal septum	Chondro-Gide^®(Geistlich Pharma AG, Wolhusen, Switzerland)	4 W	12 M FU	Human trial, 5 pt	First-in-human trial reconstruction alar lobe Autologous serum, bFGF, TGF-β1 and insulin Bilayered matrix: made of porcine-origin collagen type I and type III permeable layer and solid layer Transformation of the construct owing to environmental factors
Gentile et al. (2016)⁴²	Autologous chondrocytes	PRP mixed with chondrocytes micrografts	1 W	12 M	Human trial	Rhinoplasty Injection in the nasal dorsum
Tan et al. (2017)³⁷	Autologous keratinocytes and BMSCs	Porcine acellularized dermis matrix	None	13 M	Compassionate patient	Trachea reconstruction, lung cancer patient In vivo bioreactor, intraoperative cell seeding Nitinol stent, unclear for how long
Elliott et al. (2017)³²	Autologous BMSCs P4 and nasal epithelial cells	Decellularized homograft	48 h	15 D	Compassionate pediatric patient	Trachea reconstruction Fatal ventilatory compromise after 3 weeks Bioreactor for cell seeding
Hoshi et al. (2017)^45,46	Autologous auricular chondrocytes	PLLA	Short (n.m.)	3 Y	Human trial	Secondary adjustment cleft lip nose Dome-shaped scaffold: 3 mm thickness Substantial cartilage deposition in vivo: sustainable reconstruction Results only 3D data Confirmatory clinical trial with long-term outcomes is being developed
Zhou et al. (2018)⁴⁷	Human autologous auricular chondrocytes P2	PCL core coated with PGA-PLA layers	3 M	2, 5 Y FU	Human study (5 microtia patients)	Ear-shaped construct Microtia chondrocytes < neural crest: enhanced potency Expansion medium TGF-β1 and IGF-1 3M precultivation strategy PCL inner core for strength

The in vitro cell-scaffold 3D culture.

BM-MNC, bone marrow–mononuclear cell; Y, years; M, months; W, weeks; D, days; pt, patients; FU, follow-up; hrEPO, human recombinant erythropoietin; TGF-β1, transforming growth factor β1; G-CSF, granulocyte colony-stimulating factor; MSC, mesenchymal stem cell; PRP, platelet-rich plasma; BMSC, bone marrow stem cell; P, passage; n.m., not mentioned; PLLA, poly l-lactic acid; 3D, three dimensional; PCL, polycaprolactone; PGA-PLA, polyglycolic acid-polylactic acid; IGF-1, insulin-like growth factor 1.

Table 2.

A Glance of Clinical Studies Using Tissue-Engineered Constructs for Articular Cartilage Reconstruction

First author, Year	Cell source	Scaffold	Study type	Time in vitro^*	Time in vivo	Model	3D?	Observations
Selmi et al. (2008).⁹⁸	Autologous articular chondrocytes	Cartipatch^® (TBF Tissue Engineering; Mions, France) = agarose-alginate scaffold	Prospective phase II clinical trial	n.m.	2 Y	Human model, 17 pt	Full-thickness cartilage defect	Lesions deeper and >3 cm² improved more than smaller lesions Hyaline cartilage-like repair tissue in 50% Second generation ACI
Nehrer et al. (2008)⁹⁹	Autologous articular chondrocytes	BioCart™II (ProChon BioTech Ltd., Ness Ziona, Israel) = fibrin-hyaluronan matrix	Prospective phase II clinical trial	1 M	1 Y	Human model, 8 pt	Sponge-like 3D structure	Good filling of the defect, significant clinical enhancement after 1 Y FGF added to chondrocyte culture High seeding efficacy and enables the preservation of chondrocytic phenotype. Similar trend in patients with previous knee operation Second-generation ACI
Kon et al. (2009)¹⁰⁰	Autologous articular chondrocytes	Hyalograft^®C (Fidia, Abano Terme, Italy) = hyaluronan based	Prospective, comparative, nonrandomized trial	n.m.	5 Y	Human model, 40 pt	Full-thickness cartilage defect	Clinical superiority and sustainability of hyalograft over microfracture in large defects Cartilage resurfacing with hyaline-like maturation and stability of the implant Long-term clinical outcomes suggest lasting improvements in pain and mobility First MACT introduced in clinical practice, but withdrawal of the clinical application
Zeifang et al. (2010)¹⁰¹	Autologous articular chondrocytes	BioSeed^®-C (BioTissue Technologies, Freiburg, Germany) = PGA/PLA and polydioxanone	Randomized controlled trial	3–6 D	2 Y	Human model, 21 pt	Full-thickness cartilage defect	ACI vs. MACT, symptom improvements in both groups Excellent stability in patients with osteoarthritic degeneration No difference in efficacy, some scores favoring ACI, increased reoperation ratio in Bioseed Good clinical outcomes, persisting strength deficit, lower rate of graft failure
Schneider et al. (2011)¹⁰²	Autolgous articular chondrocytes	CaReS = collagen type 1 hydrogel	Prospective multicenter investigation	13–15 D	5 Y	Human model, 116		Good subjective outcome efficiency compared with conventional ACICaReS = Cartilage Regeneration System = second-generation ACI
Clavé et al. (2016)¹⁰³	Autologous articular chondrocytes	Cartipatch^® (TBF Tissue Engineering, Mions, France)	Multicenter RCT	n.m.	2 Y	Human model, 53 pt	Full-thickness cartilage defect	Vs mosaicplasty, both treatments showed clinical improvementCompared with microfracture, Cartipatch has better long-term outcome scores but slower postoperative recovery timeSlowest repair in the center, functional outcomes worse after cartipatchImprovement of functional outcome parameters and histological evaluation after 2 years .Third-generation ACI
Zak et al. (2014).¹⁰⁴	Autologous articular chondrocytes	Novocart^® (TETEC AG, Reutlingen, Germany) = bilayered collagen type 1 sponge with chondroitin sulfate	Prospective cases series	n.m.	2 Y	Human model, 23 pt	Full-depth cartilage cylinders	No correlation between clinical and MRI outcomesLamination of the transplants seen on MRIRight treatment of large focal chondral and osteochondral defects (midterm results)Ongoing phase III clinical trial (NCT01656902)Second-generation ACI
Mumme et al. (2016)⁶⁰	Autologous nasal septal chondrocytes	Chondro-Gide^® (Geistlich Pharma AG, Wolhusen, Switzerland) = Bilayer collagen matrix	Phase 1 study	2 W	24 M	Human model, 10 pt	Full-thickness cartilage defect	Feasible and safe useOngoing phase 2 multicenter clinical study (NCT02673905)Superiority of nasal chondrocytes to articular chondrocytes is shown in vitro. No control group in this clinical trial
Anderson et al. (2017)¹⁰⁵	Autologous articular chondrocytes	NeoCart^® (Histogenetics Corp., Waltham, MA) = bovine type 1 collagen scaffold	Prospective phase II, FDA trial	n.m.	60 M	Human model, 29 pt	Full-thickness cartilage defect	Clinical and MOCART scoresExcellent results in comparison with older cell engineering techniquesPhase I: better function, pain reduction, stability, verified as a safe procedureOngoing 249 patient Phase III clinical trial (NCT01066702) for FDA approvalThird-generation ACI

The in vitro cell-scaffold 3D culture.

ACI, autologous chondrocyte implantation; MACT, matrix-associated chondrocyte transplantation; Y, years; M, months; FU, follow-up; n.m., not mentioned; pt, patients; RCT, randomized controlled trial.

Table 3.

Status of Translational Investigation in Urology Tissue Engineering

First author, Year	Cell source	Scaffold	Study type	Time in vitro^*	Time in vivo	Model	3D?	Observations
Atala et al. (2006)⁶¹	Autologous urothelium and muscle cells	Collagen matrix > decellularized bladder submucosa OR PGA matrix	Clinical trial (cases)	7 W	3 Y	Human model, 7 pt	2 mm thick	Cystoplasty2 protocols, PGA promotes cell survival and expansion, optimal scaffold <-> homologous decellularized bladderOmentum wrap to support vascularizationPostoperative cycling to boost restoration and expansion of the implant.Fibrosis, no increase in compliance and volume
Bhargava et al. (2008)⁶²	Autologous buccal mucosa cells (keratinocytes and fibroblasts)	Human donor de-epidermized dermis	Clinical trial (cases)	7–10 D	9 Y	Human model, 5 pt	On-lay patch urethroplasty	Early take of the graft 100%Fibrosis and contraction 2/5Anastomotic stricture 3/5
Raya-Rivera et al. (2011)⁶³	Autologous muscle and epithelial cells	PGA scaffolds	Clinical trial (cases)	4–7 W	36–76 M	Human model, 5 boys	Tubularized urethroplasty	Urethroplasty in pediatric patientsComplex posterior urethral defectsTubularized engineered grafts similar histology and function to native tissue
Fossum et al. (2012)⁶⁴	Autologous urethral cells	Allogenic acellular dermis	Clinical trial (cases)	3 W	6–8 Y	Human model, 6 pt	On-lay hypospadias repair	Perineal hypospadias repairLong-term outcomesNo control group, small study population
Ram-Liebig (2012)⁶⁵	Autologous buccal cells	MukoCell^®(Bio-Medizin Zentrum Dortmund, Germany)	Phase I clinical trial	3 W	3 W	Human model, 10 pt	On-lay or inlay patch urethroplasty	Mucocell^® on the market in Germany (is an industrial product) as a scaffold for TE and to reconstruct the urethraUrethral stricture treatment5/10 impermeable and wide anastomosisMucocell^® only stable for 48 h of transportation
Joseph et al. (2014)⁶⁶	Autologous urothelial and smooth muscle cells	PGA-PLA matrix (Tengion^®; NC)	Phase II prospective study	n.m.	36 M	Human model, 11 children	Augmentation cystoplasty: partially spherical constructs	Omentum around the construct for vascularizationBladder cycling to promote expansionNo advancement in compliance and capacity, serious side effects
Bivalacqua et al. (2014)⁶⁷	Autologous dipose-derived SMCs	PLGA scaffold (Tengion^®;NC)	Phase I clinical trial	4 W	12 M	Human model, 9 pt	Urinary diversion	Phase I first in human trial compared with porcineOmentum or peritoneum around the construct for vascularization7 W postop first stage urinary tissue → 10W natural urothelium and organized tunica muscularis with ingrowth of nerve fibersOngoing multicenter phase 1 clinical trial
Ram-Liebig et al. (2017)⁶⁸	Autologous buccal mucosa cells	MukoCell^®(Bio-Medizin Zentrum Dortmund, Germany)	Multicenter prospective observational trial	1 W	24 M	Human model, 99 pt	Anterior on-lay urethroplasty	Safety in routine clinical practice, feasibility and effectiveness of the two-step procedure in human patients under real-world circumstancesHigh-quality 5-year follow-up dataLack of a control group
Barbagli et al. (2018)⁶⁹	Autologous buccal mucosa cells	MukoCell^®(Bio-Medizin Zentrum Dortmund, Germany)	Retrospective multicenter study	n.m.	55 M	Human model, 38 pt	Anterior on-lay urethroplasty	Anterior urethroplasty32/38 successClinical outcomes and accomplishments similar to standard techniques of 1-stage urethroplasty with buccal graftsLack of a control group

The in vitro cell-scaffold 3D culture.

Y, years; M, months; W, weeks; D, days; FU, follow-up; pt, patients; SMC, smooth muscle cells.

Table 4.

Status of Translational Investigation in Cardiovascular Tissue Engineering

First author, Year	Cell source	Scaffold	Study type	Time in vitro^*	Time in vivo	Model	3D?	Observations
Shin'oka et al. (2001)⁷⁰	Autologous endothelial cells (< vein)	PCL-PLA scaffold reinforced + PLA	Case report: compassionate patient	10 D	7 M	Human model, 4 year-old girl	Bypass reconstruction pulmonary artery	Pulmonary artery reconstructionDegradation after 8 W7 M good clinical and radiological resultsTime-consuming and costly cell culture, inability to use in emergency
Naito et al. (2003)⁷¹	Autologous endothelial cells (< vein)	PCL-PLA scaffold reinforced + PLA	Case report: compassionate patient	10 D	4 M	Human model, 12-year-old boy	1-mm-thick, tubular graft for bypass	Extracardiac Fontan operation, bypass between hepatic vein and main pulmonary arteryDegradation after 8 W4 M postop viable graftTime-consuming and costly cell culture, inability to use in emergency
Shin'oka et al. (2005)⁷²Hibino et al. (2010)³³Sugiura et al. (2018)⁷⁵	AutologousBM-MNC → immediate seeding and implantation on the day of operationAutologousBM-MNCAutologousBM-MNC	l-lactide with ɛ-caprolactone + PLLA	Clinical trial	Immediate implantation	1, 3 − 32 M5, 8 Y11, 1 Y	Human model, 42 pt	0.6–0-7 mm thick23 tubular grafts for extracardiac total cavopulmonary connection(19 patch)	Can be used in emergency setting. Anticoagulation 3–6 MNo calcification, no neoplasm formation, low-pressure systemFeasible and safe technique in pediatric cohort, based on experimental modelComplete endothelialization expected after 2 weeksClinical and radiographical results: no aneurysm, no rupture, no infection1 mural thrombosis, 6 graft stenosis7 asymptomatic stenosis > anastomosis tissue overgrowth, no bone formation, malignant formation, aneurysmal formation, graft infection, or rupture
Chachques , 2007⁷⁶	Autologous BMC	Type 1 collagen matrix	Phase I nonrandomized trial	4 h	10 M	Human model, 10 pt	6 mm patch	Functional recovery of the myocardium
Olausson et al. (2012)⁷⁸	Autologous endothelial and smooth muscle cells	Decellularized allogeneic donor iliac vein	Case report: compassionate patient	2 W	1 Y	Human model, 10-year-old girl	Bypass	Extrahepatic portal vein obstruction → conduit for a meso Rex bypass procedure: anastomose superior mesenteric vein and intrahepatic left portal veinReendothelialization reduces thrombogenicity and is crucial for effective patency and working vein valvesNarrowing of the construct → second graft implanted

The in vitro cell-scaffold 3D culture.

Y, years; M, months; W, weeks; D, days; FU, follow–up; pt, patients.

Table 5.

Status of Translational Investigation in Tissue-Engineered Skin and Mucosa

First author, Year	Cell source	Scaffold	Study type	Time in vitro^*	Time in vivo	Model	3D?	Observations
Pini-Prato (2003)⁷⁹	Autologous fibroblasts	Hyaluronic acid graft	Clinical trial (cases)	10 D	3 M	Human model, 6 pt	0.1 cm thickness	Gingival augmentationTotally keratinized tissue 3 M after surgeryFibroblasts positive effect on tissue restoration: boost basal connective tissue
Mohammadi et al. (2007)⁸⁰	Autologous fibroblasts	Bovine skin collagen type I	RCT	8 D	3 M	Human model, 9 pt	Partial thickness	Gingival augmentationKeratinization of the tissue after 3 MCovered with gauze and aluminum foil
Murata et al. (2008)⁸¹	Autologous fibroblasts	Hyaluronic acid matrix	Clinical trial (cases)	n.m.	30 W	Human model, 4 pt	Partial thickness	Root coverageCoronally positioned flap to cover the implant
Jhaveri et al. (2010)⁸²	Autologous fibroblasts	Acellular dermal matrix	RCT	8 D	6 M	Human model, 10 pt	Partial thickness	Gingival augmentationEsthetic concerns were objectively examinedCoronally positioned flap to cover the implantPostoperatively less inflammation
Peña et al. (2012)⁸³	Autologous immature keratinocytes	Autologous plasma-based and fibroblast scaffold	Clinical trial (cases)	n.m.	1 M	Human model, 4 pt	Full thickness	Defect of oral mucosaClinical and functional progress3 pt with splint coverage
Milinkovic et al. (2015)⁸⁶	Autologous fibroblasts	Bilayer collagen membrane (Bio-Gide^®: Geistlich Biomaterials, Wolhausen, Switzerland)	RCT	2 D	12 M	Human model, 18 pt	Bilayer membrane	Gingival augmentationPositive healing features, decreased operative time and patient morbiditySignificant progress, reliable and successful treatmentVS connective tissue graft: similar resultsCoronally advanced flap to cover
Llames et al. (2014)⁸⁵	Autologous immature keratinocytes	Autologous plasma-based and fibroblast scaffold	Compassionate patient	n.m.	2 Y	Human model, pediatric patient	Full thickness	Gingival augmentationInexpensive model of full-thickness tissue-engineered mucosaOff-the-shelf in 3 W
Sieira et al. (2015)⁸⁷	Autologous keratinocytes	Plasma-based scaffold full of autologous fibroblasts	Case reports	Immediately	4 M	Human model, 2 cases	Full thickness	Tissue-engineered keratinized oral mucosa, resistant to friction and stable <-> muscle/skin flapNo donor site morbidity, less postsurgical pain and risk of infectionMaxilla and mandibular reconstructionOne case: Prelaminating the fibula flapOther case: second-stage grating of the fibula after mandibular reconstruction

The in vitro cell-scaffold 3D culture.

Y, years; M, months; W, weeks; D, days; FU, follow-up; n.m., not mentioned; pt, patients.

Discussion

There are four essential elements of any TE approach to reconstruct a tissue or organ: a matrix, a cell source, mechanical and biochemical signals that enable new tissue formation, and for most of the tissues also the identification of a source for blood supply. The design protocols used to incorporate these four elements differ considerably in different organs and have to be adapted to specifically to the organ that is being replicated.

In addition, the standard TE technique has two distinct phases in a clinical setting: in vitro cultivation of 3D cell scaffolds, and the in vivo development and integration of the tissue-engineered medical construct in the real-time clinical wound environment.

Most translational advances of tissue-engineered medical products were reported in the reconstruction of elastic cartilage in reconstruction protocols of nose, ear, and trachea followed by studies with tissue-engineered articular cartilage. Clinical tissue-engineered constructs were also reported for the treatment of urological defects, cardiovascular defects, and mucosal defects.

Clinical Use of TE Elastic Cartilage

There is a substantial difference between the morphology and requirements of elastic and articular cartilage.¹⁶ Large laboratory animal studies reporting tissue-engineered cartilage constructs were critically analyzed, and promising and challenging outcomes were identified (Table 6). TE constructs undergo degeneration, inflammation, fibrosis, and foreign body reaction after implantation.^17–22 Furthermore, a critical factor that hinders translation is the worsened vascularization of large structures that initiates necrosis and poor cell expansion.²³ Cartilage tissue itself is avascular but it is enclosed by well-developed vascular networks. Preclinical models have shown that implanted tissue-engineered constructs obtain their nutrients primarily by tissue fluid permeation and secondarily by neovascularization originating from the enclosed tissues.^24–26 The central area of the construct experiences ischemia at the beginning of transplantation, which triggers revascularization. This process takes multiple weeks. Therefore, a substantial part of seeded cells may not survive and will participate instead in the degradation of the construct.

Table 6.

Status of Large Animal Studies in Plastic Reconstructive Tissue Engineering

First author, Year	Cell source	Scaffold	Tissue	Time in vitro^*	Time in vivo	Model	3D?	Observations
Cao et al. (1998)¹⁷	Autologous auricular chondrocytes	Pluronic F-127PGA scaffoldCalcium alginate	Ear	n.m.	6 W	Porcine model	3D	No ear-shaped constructPluronic constructs developed into elastic cartilagePGA and calcium alginate scaffolds resulted in inflammation and heavy fibrotic reaction
Chang et al. 2003¹⁰⁶	Autologous auricular chondrocytes	Alginate hydrogel	Ear	n.m.	30 W	Ovine model	3D	Nose bridge and chin implant shapeSubcutaneously implantation in the dorsal neck
Xia et al. 2004¹⁸	Autologous MSCs	Chitosan–gelatin complex	Ear	n.m.	10 and 16 W	Porcine model	2-mm-thick sheet	No ear-shaped constructHistological, mechanical and biochemical properties similar to native elastic cartilage.Engineered cartilage was not homogenous
Rotter et al. (2005)¹⁰⁷	Auricular chondrocytes	PGA-PLA (Ethisorb)	Ear	1 W	1, 4 and 8 W	Porcine model	3D disk construct	No ear-shapedIL-1α in the vicinity of degrading polymer.GAG content and cartilage formation higher in serum-free cultured implants.Cartilage formation after 1 W implantation
Go et al. (2010)¹⁹	Autologous MSC-derived chondrocytes and epithelial cells	Decellularized homograft	Trachea	n.m	60 D	Porcine model	Trachea model	Chondrocyte and epithelial cell proliferation are essential for graft survivalBioreactor for cell seeding
Morotomi et al. (2012)¹⁰⁸	Autologous auricular chondrocytes P1	PGA + fibrin + bFGF-gelatin complex	Ear	24 h	1, 3, 5, and 10 W	Canine model	150-μm-thick sheet	No ear-shaped constructFibrin-PGA complex as ideal scaffoldbFGF as growth- and angiogenesis-inducing factorInterfascial implant superior to subcutaneous
Asawa et al. (2012)²⁰	Autologous auricular chondrocytes	PGA or PLLA + atelocollagen hydrogel	Ear	n.m.	1, 2 and 6 M	Canine model	0.3-cm-thick sheets	No ear-shaped constructPLLA > > PGA in cartilage production and shape preservationPGA > > PLLA in biodegradation
Kanazawa et al. (2013)²¹	Autologous auricular chondrocytes	Agarose nonabsorbable hydrogel	Ear	n.m.	1 M	Beagle model	3-mm-thick sheets	No ear-shaped constructComplete resorption accompanied by inflammation. No cartilage formation in canine model as was seen in mouse model
Bichara et al. (2014)²³	Autologous auricular chondrocytes	Fibrous collagen scaffold	Ear	2, 6 OR 12 W	6 and 12 W	Ovine model	2 mm sheets	No ear-shaped constructSuperior cartilage after 2 W in vitro cultureFirst illustration of elastin expression in tissue-engineered auricular cartilage in a ovine model
Pomerantseva et al. (2016)⁵⁴	Autologous auricular chondrocytes	Porous collagen with titanium framework	Ear	n.m.	20 W	Ovine model	Ear-shaped construct	BFGF supplementationElastin formation in a stable cartilage engineered construct after extreme expansion of chondrocytes
Ding et al. (2016)¹⁰⁹	Bone marrow MSCs (BMSCs)	PGA-PLA scaffold	Ear	8 W	2, 4, and 8 W	Porcine model	Cartilage cylinder	No ear-shaped constructBMSC-based engineered cartilageIncreasing M2 polarization of macrophages
Liu et al. (2016)⁵⁵	Autologous auricular chondrocytes P1	PGA-PLA scaffolds	Ear	2, 8, and 12 W	1 and 8 W	Goat model	Cartilage cylinder	No ear-shaped construct
Clark et al. (2016)¹¹⁰	BM-MNCs	PET/PU scaffold	Trachea	None	6 W	Sheep model	Trachea model	Inspecting the previously used in human tissue-engineered constructVacuum-seeding technique, no in vitro cultureEpithelial migration front at anastomoses
AL-Ayoubi et al. (2017)²⁴	Human MSCs	Acellular bovine dermis extracellular matrix	Trachea	1 W	3 M	Porcine model	4-mm-thick anterior trachea segment	Anterior tracheal defect reconstructionIngrowth of native cells in the constructDevelopment of blood vessels
Pepper et al. (2017)¹¹¹	Autologous BM-MNCs	PET/PU scaffold with polycarbonate rings	Trachea	None	4 M	Sheep model	Trachea model	Management of tissue-engineered graft stenosisVacuum-seeding technique, no in vitro culture
Gilevich et al. (2018)¹¹²	Allogenic MSCs	PET scaffold	Trachea		1 M	Primate model	Trachea model	Good biocompatibilityBioreactor for cell seedingResults very briefly described
Xia et al. (2019)²⁵	Autologous auricular cartilage cells	PCL scaffold	Trachea	5 W	8–14 W	Goat model	3D printed trachea	Epithelial-tissue like formation and vascularizationGranulation of tissue hyperplasia
Li et al. (2019)²⁶	Autologous auricular cartilage cells P2 and oral mucosal epithelium	Cartilage sheet	Trachea	4 W	6 W	Goat model	4-layer cartilage sheet	Extensive cartilage regenerationStable prevascularizationMuscle-cartilage–fascia–mucosa structureGood vascularization and epithelialization in vivo (heterotopic implantation)PCL internal support tube
Pepper et al. (2019)²²	Autologous BM-MNCs	PET/PU scaffold with polycarbonate rings	Trachea	None	4 M	Lamb model	Trachea model	Investigating the trachea implant used in the failed human models

The in vitro cell-scaffold 3D culture.

Y, years; M, months; W, weeks; D, days; H, hours; FU, follow-up; bFGF, basic fibroblast growth factor; P, passage; BMSC, bone marrow stem cell; PET/PU, polyethylene terephthalate/polyurethane.

Malformation or injury of the cartilages of the ear, nose, and trachea necessitate complex surgical reconstructions to rebuild absent cartilage and bony support. The use of autologous cartilage obtained from the rib, auricle, or nasal septum is the gold standard for reconstructing craniofacial cartilage defects owing to their lack of immunogenicity.²⁷ However, disadvantages such as limited availability of graft tissue, donor site morbidity, and variable tissue quality remain. Furthermore, the inability to implant cartilage with growth potential in neonates and small children poses another limitation. In addition, rib fibrocartilage differs from flexible elastic cartilage and is subject to aging calcification.²⁸ Cartilage reconstruction with alloplastic implants is universally available as an alternative to autologous grafts.²⁹ Because of the consistent pre-established shape and straightforward surgical implantation procedure, some researchers prefer this reconstruction approach. However, the possibility of infection, lack of biocompatibility, extrusion, and unknown long-term sustainability are significant drawbacks.

TE protocols for tracheal replacement

To achieve a durable functional restoration of long-segment tracheal defects, a tissue-engineered substitute needs to have (1) extensive cartilage regeneration capability to prevent airway collapse, (2) stability, (3) effective prevascularization capacity to support cartilage viability and epithelialization, and (4) a functional epithelium to avoid infections and granulated hyperplasia. In contrast to other tissues, the purpose of TE for trachea is not to enhance malfunctioned tissue, but to replace a vital organ to save function and life.

In 2010, a 10-year-old boy with congenital tracheal stenosis and aorto-tracheal fistula received a tissue-engineered tracheal replacement.³⁰ Because of the urgent nature of the condition, the multidisciplinary team had to perform a direct protocol for the development of the construct. The epithelium was fully restored after 1 year. The question that arose was whether this epithelialization was induced by the epithelial patches or whether it originated from nearby healthy tissue. A 4-year follow-up report declared the good health of the boy. In decellularized tracheas, the reepithelialization process is extensive to preserve the construct and to save the graft from the fibroproliferative reaction of the recipient.³¹ Disadvantages of decellularized scaffolds are size matching, demanding decellularization processes, and the need for allogeneic tissues. Elliott et al. successfully implanted a cell-seeded decellularized homograft in a 15-year-old girl with critical tracheal stenosis under compassionate-use license.^30,32 According to the hypothesis that seeding a neovessel would inhibit graft stenosis,^33,34 they seeded the graft with bone marrow stem cells (BMSCs). The graft was maintained for 2 weeks. The early results were promising and increased exercise tolerance by the host until the abrupt tracheal obstruction of the posterior wall. The long-term follow-up of these patients is unclear. It seems they were eventually treated with endotracheal stents that prevented collapse of the airway.^35,36 This knowledge is essential because the use of stents elicits a strong inflammatory response, which may be a cause of hypergranulation and hence obliteration and collapse.

Tan et al.³⁷ developed an in vivo bioreactor for connecting the tissue-engineered airway construct with the external environment through two Port-a-cath^® catheters. For 1 month, the bioreactor was perfused by Ringer's gentamicin fluid to prevent infection, keep the preseeded cells alive, and allow reseeding of the scaffold with BMSCs. The authors observed the secretion of several angiogenic growth elements, which confirmed the migration of the reseeded cells to the tissue-engineered construct.

The mechanical alterations of the constructs after transplantation in human trials are very evident but were not observed in earlier pig models.¹⁹ This indicates that human trials are essential to identify the limitations and strengths of a tissue-engineered construct, as animal models cannot fully emulate the complex pathological processes in humans.¹⁹ Furthermore, patients in compassionate-use programs are not treated as study subjects, and thus allow only the most necessary procedures to be performed. Therefore, both trials with compassionate patients as well as relevant in vivo studies need to be combined to establish a clear-cut study protocol for clinical trials that meets regulatory requirements.

The long-term outcomes of this sequence of clinical trials revealed apparent drawbacks in the used techniques and require transparent research in relevant in vivo models before clinical translation. The main complication of a tissue-engineered trachea is graft stenosis, which poses a primary barrier to clinical translation. Especially for the trachea, it is imperative that the hollow convexity is stably maintained to facilitate efficient ventilation.

The most common difficulty associated with the application of autologous cartilage in the reconstruction of the trachea is the absence of respiratory epithelium and subsequent lack of mucociliary clearance that causes mucous stagnation, graft stenosis, and chondromalacia.³⁸ Moreover, as the inner respiratory ciliated epithelium of the windpipe is the first defence mechanism against aggressive particles present in air, a well-vascularized inner epithelial lining is essential in all reconstructive procedures. It is striking that none of the previous trials developed a prevascularized trachea, as sufficient blood supply is crucial for the survival of the tissue-engineered implant. These particular features of the windpipe make a TE approach quite complex.^7,12,39

For tracheal substitution, two other elementary approaches have dominated the field: tracheal allotransplantation and aortic homograft transplantation (Fig. 2). The complex capillary-based blood supply in the trachea discourages regular allotransplantation because the trachea lacks a detectable vascular pedicle. This key issue prevents efficient tracheal transplantation. Furthermore, the substitute should offer elasticity with sufficient stiffness and long-term efficacy alongside biological integrity to avoid rejection, dehiscence of the anastomosis, and weakening. Trachea transplantations with cadaveric allografts and aortic homografts fail to meet these conditions and require high-dose immunosuppression to prevent acute graft rejection owing to the antigenically active cells in the airway mucosa.^7,11 These challenges, together with the heterogeneous presentation of tracheal conditions, make the production of an adequate tracheal replacement very difficult. Potential solutions to the complications and limitations associated with the aforementioned approaches may be found with TE techniques:

FIG. 2.

Tracheal allotransplantation. Upper row: Trachea allotransplantation to restore a long-segment defect of the trachea. A donor trachea is prelaminated in the vascularized fascia of the forearm and allogenic mucosa is replaced with recipient autologous buccal mucosa. This creates a chimeric construct that consist of autologous cell lining within an allogenic cartilaginous framework. This chimera strategy generates a certain degree of “tolerance”. Microvascular transfer to the defect occurs in a second stage after weeks. Immunosuppression can be tapered and stopped after control bronchoscopy.^9,10 Lower row: currently the authors investigate in a preclinical setup the cultivation of autologous respiratory cells and gentle decellularization techniques on the allogenic trachea to diminish immunogenicity even further and to induce faster reepithelialization and angiogenesis.^11,41 Color images are available online.

Cell cultures of ciliated epithelium seeded in a porous cartilaginous matrix or a decellularized allogenic trachea and banked on vascularized fascia for transplantation after “prefabrication,” may lead in future to an authentic trachea construct.

TE protocols for replacement of nasal cartilage

Fulco et al.⁴⁰ pioneered the first human trial of TE reconstruction of the alar lobe and recommended a safe functional and esthetic replacement approach for native cartilage. In vitro fabricated extracellular matrix (ECM) with characteristics of mixed hyaline and fibrous cartilage was implanted in vivo. After 6 months, it transformed into a mixture of fibrous connective tissue with muscle fibers and fat cells similar to alar tissue. This change is attributable to the lack of environmental cues required for the preservation and progression of the cartilaginous phenotype and the availability of other cues for the conversion of the cartilage that corresponded to the tissue at the site where it had been placed.

Given that platelet-rich plasma (PRP) is biocompatible and contains several growth factors, such as transforming growth factor β1 (TGF-β1), fibroblast growth factor 2 (FGF-2), and insulin growth factor 1 (IGF-1), it is a propitious cell carrier for TE. Based on successful results in recently published reports, Gentile et al.^41,42 created a cartilaginous construct made of nasal chondrocyte micrografts mixed with PRP for subcutaneous injection in the nasal dorsum for the nasal alar reconstruction in 11 patients. They combined PRP with chondrocyte micrografts to prevent rapid contraction and degradation in vivo. In our experience, PRP neither maintains shape nor volume and therefore cannot serve as a 3D matrix that guides cells into the desired shape.⁹ However, it induces angiogenesis and promotes healing by proangiogenic growth factor release,^43,44 and can be used for other purposes.

The exploratory first-in-human clinical trial of Hoshi et al.^45,46 indicated that the application of autologous tissue-engineered cartilage with poly-l-lactic acid as a matrix was secure for the secondary adjustment of the cleft lip-nose in three patients. Unfortunately, their report lacked histological and immunohistochemical evidence for the implanted cartilage, and the results only relied on the 3D data from computed tomography and magnetic resonance imaging of the glycosaminoglycan content. The association between the imaging and evidence from histopathologic research still needs to be determined before making substantial conclusions.

In contrast to the research conducted on other tissues, there is an insufficient number of high-quality reports concerning TE strategies in rhinology. Apart from these three human studies, there are no reports on nasal TE in large animals.

TE protocols for replacement of auricular cartilage

Thirty years after the landmark study of Vacanti et al.,³ the first clinical trial of a tissue-engineered ear implant was published. The bench-to-bedside study described by Zhou et al.⁴⁷ is a pilot clinical trial for the reconstruction of the human ear with a human ear-shaped cartilage engineered in vitro. At present, the longest follow-up period described in this trial is 2.5 years. This certainly is a landmark study. The authors used autologous chondrocytes obtained from the remains of the microtia ear cartilage of the patients as the seed cell source. They used a prolonged in vitro precultivation period to diminish the postimplantation inflammatory response. The investigators used computer-aided design and computer-aided manufacturing to generate a patient-specific poly-glycolic-PLA scaffold for cartilage seeding, with a polycaprolactone center to ensure the mechanical strength of the scaffold. The five patients are currently in follow-up.

Translational hurdles for clinical application of TE elastic cartilage

The ideal scaffold is a nonimmunogenic, long-lasting, biocompatible construct with growth potential. This construct ideally must be individualized for instance with the help of CAD-CAM and 3D printing, making it applicable for the morphological and structural requirements of the recipient (see Fig. 3). Both preclinical and clinical outcomes indicate the difficulty of this mission. Biosynthetic scaffolds were synthesized to overcome the inherent limitations of decellularized scaffolds: they are commercially available, easily adjustable, and physiologically neutral. However, biocompatibility, immune response, and an unknown long-term fate of the scaffold in cartilage TE still are substantial obstacles that require further research.⁴⁸

FIG. 3.

Clinical tissue engineering strategies. 1—(upper left) CT scan data of the required matrix are translated in DICOM format to a Virtual Surgical Planning software package where the required custom made matrix is designed. 2—After Computer-Aided Design (CAD), the 3D matrix subsequently is 3D printed by Computer-Aided Manufacturing. 3—(left) From autologous biopsies of the patient, cell populations are cultured within the matrix directly or ex vivo and transferred to the matrix in second stage. 4—(below) Proangiogenic growth factors and endothelial progenitor cells are integrated in the incubation environment to generate blood vessel networks. 5—(right) After generation of macroscopic blood vessels in the matrix, the 3D custom-made implant can be transferred into the clinical situation by microvascular transfer. Nb. Quasiavascular tissues such as “custom-made and shaped” cartilage and bone do not need steps 4 and 5. Color images are available online.

Second, auricular, septal, alar, and tracheal cartilage, have a shared embryonic origin, but display different cartilage subtypes. Following new evidence, the choice of the cell source is one of the most important factors for successful cell therapy.^49–51 Chondroprogenitors found in articular cartilage and the perichondral structure of auricular cartilage are now considered as superior cellular components.⁵² Adult autologous cells bear the disadvantage of poor growth and dedifferentiation potential in vitro.

For tissue reconstruction, cells are expanded in vitro to mature to secure an extensive cell population. Chondrocytes grown in monolayer cultures do not fully regain their original phenotype,²³ but the use of 3D structures for chondrocyte culture has been shown to maintain the differentiated chondrocyte phenotype. A highly chondrogenic microenvironment with growth factors and accurate cartilage ECM molecules is fundamental in assuring sufficient chondrogenesis and in the prevention of dedifferentiation.⁵³ Other reasons for TE failure include independent cell sources that cause overgrowth of immature new cartilages, which are likely to break and stiffen, eventually resulting in calcification.⁵⁴ Multidisciplinary groups are collaborating to improve the bioreactor technology for emulating the required conditions under which tissues can be engineered.

The implanted biodegradable cartilage scaffold with neocartilage formation can induce an active inflammatory response, which adversely affects chondrogenesis and encourages the resorption of autologous neocartilage. Inflammation is primarily induced by the degradation of biomaterials or scaffolds and elicits an immune reaction regardless of whether scaffolds are synthesized with biological or artificial biomaterials. The degree of maturation of the cultured chondrocytes also influences this immune reaction. Scientists have introduced “in vitro precultivation” to create mature cartilaginous tissue with improved neocartilage properties and the ability for an appropriate breakdown of scaffold materials and matrix accumulation before implantation.⁵⁵

Clinical TE Trials Other Than (Elastic) Cartilage

Tables 2–5 give an overview of clinical studies on TE principles and substitutes for the treatment of articular cartilage breakdown,^56–60 urological defects,^61–69 cardiovascular frailty,^33,70–78 and mucosal defects.^79–87

Clinical TE trials for articular cartilage

Matrix-induced autologous chondrocyte transplantation (MACT) is a technique used for the reconstruction of full-thickness articular cartilage defects and consists of a 3D cell carrier seeded with cultured autologous cells. Bone marrow mesenchymal stem cells are widely investigated in articular cartilage TE trials.^88–90 Besides their immunoprivileged and immunomodulatory capabilities that make them convenient for application in inflammatory diseases, they also have a striking ability for multiplication and chondrogenesis. However, a major obstacle is the initiation of calcification and hypertrophy.⁹¹ On the contrary, multiple studies have tried to culture mature chondrocytes, but owing to limited quantities of obtained chondrocytes, their utilization in clinical trials has been limited. However, growth factors and exogenous stimuli are proved to be a very important factor for the production of hyaline-like cartilage. Nasal chondrocytes, in contrast, are declared in some research protocols to be superior to articular chondrocytes with the production of higher quality hyaline cartilage.^40,60

Given the numerous clinical studies describing articular TE, this review briefly lists some to compare different cell carriers (Table 2).

The results from the first phase III clinical study of an articular construct will soon be published to gain Food and Drug Administration (FDA) approval for the NeoCart^® construct (NCT01066702). Mumme et al.⁶⁰ developed another technique subsequent to the publication of the results of the human trial that was used to rebuild the alar lobe with autologous nasal chondrocyte constructs.⁴⁰ They seeded nasal septum chondrocytes (Chondro-Gide^®) for the reconstruction of articular cartilage defects in a phase I study with satisfactory outcomes resulting in the ongoing phase II multicenter clinical study (NCT02673905). Owing to the different primary endpoints and scoring scales, it is difficult to compare this novel MACT technique with existing methods and to demonstrate its superiority.

Clinical TE trials for bladder and urethra

The ultimate target of generating a long-lasting, functional tissue-engineered duct, as was expected after the achievement of the first in-human tissue-engineered cystoplasty 20 years ago by Atala et al.,⁶¹ has not been achieved yet. However, substantial technical and preliminary steps have been taken (Table 2). Phases I and II of a clinical trial achieved success in bladder substitution with a bioresorbable scaffold (Tengion^®).^66,67 A multicenter phase I clinical trial is currently being developed (NCT01087697). It should be noted that despite the progress in the field, the substitutes are very thin and do not have any functional features resembling those of a natural bladder. This lack of functionality and original shape is even more important in the case of the ureter. Almost all the substitutes produced so far have been developed for transplant in an onlay manner and only have partial thickness.^61–69 Nonetheless, Raya-Rivera et al.⁶³ reported satisfactory long-term results of tubularized urethroplasty in five boys with complex posterior urethral defects. The grafts were composed of autologous muscle cells and epithelial cells seeded on PGA scaffolds.

Clinical TE trials for heart valves and myocardial tissue

The application of tissue-engineered heart valves with cell-seeded scaffolds in clinical practice has been suspended because of the unscalable nature, time-intensiveness, and high cost of the process, as well as its unavailability for emergencies.⁹² However, TE approaches can be used for the development of biologically based ducts to address the need for vascular grafts (Table 4). The largest trial was an 11-year follow-up study in 42 patients.⁷² The recurrence of asymptomatic vascular restenosis was the principal obstacle and was associated with tissue overgrowth at the suture site, for which the patients were treated with balloon angioplasty.⁹³ Seeding higher amounts of BMSCs on the graft and adding TGF-β to the medium is a potential realistic approach to supporting graft patency.

The electrical conduction and powerful contractile force of myocardial tissue make its substitution by TE extremely challenging. There have been proposals for the use of scaffold-free cell sheets, acellular grafts, and bioengineered recellularized scaffolds, but the literature includes only one TE trial.^76,77 Chachques et al.⁷⁷ implanted a cell-seeded, biodegradable, 6-mm-thick 3D collagen type I patch in the epicardia of 10 patients with postischemic myocardial lesions. Although this has been described as a superior technique, no subsequent studies were conducted after this 2008 report.

Clinical TE for skin and mucosa

A substantial number of epidermal and dermal substitutes are commercially available and have been successfully applied in clinical practice. In burn wound care, dermal substitutes include a base layer that enhances the elasticity and pliability of the overlying thin skin graft. However, these substitutes only represent the superficial layer of the skin and do not incorporate hair follicles, apocrine glands, or sensory nerve buds.^14,94 Such substitutes do not meet the criteria for TE.⁴³ However, for gingival augmentation and oral mucosa reconstruction, several case series studies have achieved a cell-seeded matrix (Table 5)^79–87 and full-thickness mucosa. Long-term studies using larger sample sizes are essential to concluding the superiority of tissue-engineered gingival mucosa over currently used techniques such as grafting of autologous buccal mucosa.

Undoubtedly, a TE strategy for achieving full-thickness skin including the aforementioned elements is one of the most anticipated and clinically required therapeutic innovations.

Additional hurdles toward translation of TE medical products

There are numerous additional obstacles for the transition of TE medical products toward clinical practice. In first stage there is a critical need for early transdisciplinary collaboration and a shortage of sustained funding programs for multidisciplinary translational research.

Furthermore, large number of ventures fail to attract or retain investor funding, promotion, and clinical acceptance. Investigators often fail to accurately identify critical clinical adoption criteria because of their focus of improved patient outcomes. Other criteria such as ethical concerns, price, alternatives, and place in the workflow can result in a failure of the product.^95–97

Moreover, market entry requires elaborate and expensive clinical trials to deliver high-quality clinical evidence analyzing safety, efficacy, and accuracy of the product. More than 80% of all clinical trials fail to result in Federal Drug Administration approval. By utilizing a bedside to bench and back again approach, investigators can improve the odds that their research will have a meaningful clinical impact.

Limitations of this review

Several important limitations are associated with this study and need to be acknowledged. The first limitation is the narrative nature of this study with its inherent subjectivity. We switched the focus from the comparison of the outcomes of published clinical studies to a conclusive reflection of the development and innovation of TE in clinical studies that have been published. Second, owing to the paucity of randomized controlled trials, many of the included studies were case reports and articles that described outcomes in compassionate patients. The evidence in these studies is minor; however, these studies are worth mentioning because the literature only offers a few high-quality studies that have examined TE in humans.

Conclusion

It is critical to determine the cues that are mandatory to preserve the stability of the regenerated tissue and ascertain if it is really possible to generate a tissue-engineered construct that can stimulate its growth and expansion. Additional important questions that need to be addressed include the determination of the angiogenic mechanisms that could improve neovascularization in the implanted constructs and whether use of detergents to extract cells from tissue biopsy is truly safe. These are only a few of the many important questions that further research should address in the domain of TE to deal with remarkable challenges and achieve progress in clinical practice.

This review listed individual strategies within the field of TE that have been published. To date, only two published clinical studies have reported functional, 3D tissue-engineered constructs.^40,47 Accordingly, the need to combine diverse domains of expertise to facilitate the creation of a tissue-engineered organ for clinical application is emphasized. The diversity of procedures reported in the literature indicates that a durable tissue-engineered approach remains unavailable. These are the initial stages of what will subsequently evolve in a feasible, free-graft substitute for regenerative surgery in the future. Each model must be exposed to a thorough investigation along with the exploration of molecular and cellular processes that support new tissue generation. Merely mixing fundamental TE materials will not prove satisfactory for the accurate clinical translation of tissue-engineered constructs. It is of paramount importance that advancements in preclinical and clinical outcomes are extensively reported within predetermined metrics to homogenize information to enable protocol interventions and to assess procedural cost-effectiveness. There is a critical need for early transdisciplinary collaboration and sustained funding programs for multidisciplinary translational research.

Footnotes

Authorship Confirmation Statement

All co-authors have reviewed and approved the article before submission.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Vogel

Report finds trachea surgeon committed misconduct. Science (80-) 2015 [Epub ahead of print]; DOI:10.1126/science.aac4623.

Langer

, and Vacanti

J.P.

Tissue engineering. Science (80-), 260, 920, 1993.

Vacanti

C.A.

, Cima

L.G.

, Ratkowski

, Upton

, and Vacanti

J.P.

Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes. MRS Proc, 252, 367, 1991.

Vacanti

C.A.

, Langer

, Schloo

, and Vacanti

J.P.

Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plast Reconstr Surg, 88, 753, 1991.

Langer

, and Vacanti

Advances in tissue engineering. J Pediatr Surg, 51, 8, 2016.

Thorsten

Tracheobronchial bio-engineering: biotechnology fulfilling unmet medical needs. Adv Drug Deliv Rev, 63, 367, 2011.

Den Hondt

, Vanaudenaerde

B.M.

, Verbeken

E.K.

, and Vranckx

J.J.

Epithelial grafting of a decellularized whole-tracheal segment: an in vivo experimental model. Interact Cardiovasc Thorac Surg, 26, 753, 2018.

Dickens

, Vermeulen

, Hendrickx

, Van den Berge

, and Vranckx

J.J.

Regulable vascular endothelial growth factor ₁₆₅ overexpression by ex vivo expanded keratinocyte cultures promotes matrix formation, angiogenesis, and healing in porcine full-thickness wounds. Tissue Eng Part A, 14, 19, 2008.

Vermeulen

, Dickens

, Degezelle

, Van den Berge

, Hendrickx

, and Vranckx

J.J.

A plasma-based biomatrix mixed with endothelial progenitor cells and keratinocytes promotes matrix formation, angiogenesis, and reepithelialization in full-thickness wounds. Tissue Eng Part A, 15, 1533, 2009.

10.

Delaere

, Vranckx

, Verleden

, De Leyn

, and Van Raemdonck

Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med, 362, 138, 2010.

11.

Delaere

P.R.

, Vranckx

J.J.

, and Den Hondt

Tracheal allograft after withdrawal of immunosuppressive therapy. N Engl J Med, 370, 1568, 2014.

12.

Vranckx

J.J.

, Den Hondt

, and Delaere

Prefabrication and prelamination strategies for the reconstruction of complex defects of trachea and larynx. J Reconstr Microsurg, 30, 145, 2014.

13.

Delaere

P.R.

, Vranckx

J.J.

, Meulemans

, et al. Learning curve in tracheal allotransplantation. Am J Transplant, 12, 2538, 2012.

14.

Vranckx, J., and Den Hondt, M. Tissue Engineering. In: Wei, Mardini

F.C.

, S., eds. Flaps and Reconstructive Surgery. 2nd edition. Toronto, Canada: Elsevier, 2017, pp. 189–202.

15.

Cao

, Vacanti

J.P.

, Paige

K.T.

, Upton

, and Vacanti

C.A.

Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg, 100, 297, 1997; discussion 303.

16.

Jessop

Z.M.

, Zhang

, Simoes

, et al. Morphological and biomechanical characterization of immature and mature nasoseptal cartilage. Sci Rep, 9, 1, 2019.

17.

Cao

, Rodriguez

, Vacanti

, Ibarra

, Arevalo

, and Vacanti

C.A.

Comparative study of the use of poly(glycolic acid), calcium alginate and pluronics in the engineering of autologous porcine cartilage. J Biomater Sci Polym Ed, 9, 475, 1998.

18.

Xia

, Liu

, Cui

, et al. Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds. J Biomed Mater Res, 71B, 373, 2004.

19.

, Jungebluth

, Baiguero

, et al. Both epithelial cells and mesenchymal stem cell–derived chondrocytes contribute to the survival of tissue-engineered airway transplants in pigs. J Thorac Cardiovasc Surg, 139, 437, 2010.

20.

Asawa

, Sakamoto

, Komura

, et al. Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model. Cell Transplant, 21, 1431, 2012.

21.

Kanazawa

, Fujihara

, Sakamoto

, et al. Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel. J Tissue Eng Regen Med, 7, 1, 2013.

22.

Pepper

, Best

C.A.

, Buckley

, et al. Factors influencing poor outcomes in synthetic tissue-engineered tracheal replacement. Otolaryngol Neck Surg, 161, 458, 2019.

23.

Bichara

D.A.

, Pomerantseva

, Zhao

, et al. Successful creation of tissue-engineered autologous auricular cartilage in an immunocompetent large animal model. Tissue Eng Part A, 20, 303, 2014.

24.

Al-Ayoubi

A.M.

, Rehmani

S.S.

, Sinclair

C.F.

, Lebovics

R.S.

, and Bhora

F.Y.

Reconstruction of anterior tracheal defects using a bioengineered graft in a porcine model. Ann Thorac Surg, 103, 381, 2017.

25.

Xia

, Jin

, Wang

, et al. Tissue&δαση;engineered trachea from a 3D-printed scaffold enhances whole-segment tracheal repair in a goat model. J Tissue Eng Regen Med, 13, 694, 2019.

26.

, Yin

, Liu

, et al. Regeneration of trachea graft with cartilage support, vascularization, and epithelization. Acta Biomater, 89, 206, 2019.

27.

Rotter

, Haisch

, and Bücheler

Cartilage and bone tissue engineering for reconstructive head and neck surgery. Eur Arch Oto-Rhino-Laryngol, 262, 539, 2005.

28.

Reighard

C.L.

, Hollister

S.J.

, and Zopf

D.A.

Auricular reconstruction from rib to 3D printing. J 3D Print Med, 2, 35, 2018.

29.

Patel

, and Brandstetter

Solid implants in facial plastic surgery: potential complications and how to prevent them. Facial Plast Surg, 32, 520, 2016.

30.

Elliott

M.J.

, De Coppi

, Speggiorin

, et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet, 380, 994, 2012.

31.

Genden

E.M.

, Iskander

, Bromberg

J.S.

, and Mayer

The kinetics and pattern of tracheal allograft re-epithelialization. Am J Respir Cell Mol Biol, 28, 673, 2003.

32.

Elliott

M.J.

, Butler

C.R.

, Varanou-Jenkins

, et al. Tracheal replacement therapy with a stem cell-seeded graft: lessons from compassionate use application of a GMP-compliant tissue-engineered medicine. Stem Cells Transl Med, 6, 1458, 2017.

33.

Hibino

, Mcgillicuddy

, Matsumura

, et al. Late-term results of tissue-engineered vascular grafts in humans. J Thorac Cardiovasc Surg, 139, 431, 2010.

34.

Hibino

, Yi

, Duncan

D.R.

, et al. A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts. FASEB J, 25, 4253, 2011.

35.

Hamilton

N.J.

, Kanani

, Roebuck

D.J.

, et al. Tissue-Engineered Tracheal Replacement in a Child: A 4-Year Follow-Up Study. Am J Transplant, 15, 2750, 2015.

36.

Delaere

P.R.

, and Raemdonck

Van. Commentary: the sobering truth about tracheal regeneration. J Thorac Cardiovasc Surg, 159, 2537, 2019.

37.

Tan

, Liu

, Chen

, et al. Clinic application of tissue engineered bronchus for lung cancer treatment. J Thorac Dis, 9, 22, 2017.

38.

Fabre

, Kolb

, Fadel

, et al. Successful tracheal replacement in humans using autologous tissues: an 8-year experience. Ann Thorac Surg, 96, 1146, 2013.

39.

Den Hondt

, Vanaudenaerde

, Verbeken

, and Vranckx

J.J.

Requirements for successful trachea transplantation: a study in the rabbit model. Plast Reconstr Surg, 141, 845e, 2018.

40.

Fulco

, Miot

, Haug

M.D.

, et al. Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial. Lancet (London, England), 384, 337, 2014.

41.

Scioli

M.G.

, Bielli

, Gentile

, Cervelli

, and Orlandi

Combined treatment with platelet-rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose-derived stem cells in three-dimensional collagen scaffolds. J Tissue Eng Regen Med, 11, 2398, 2017.

42.

Gentile

, Scioli

M.G.

, Bielli

, Orlandi

, and Cervelli

Reconstruction of alar nasal cartilage defects using a tissue engineering technique based on a combined use of autologous chondrocyte micrografts and platelet-rich plasma: preliminary clinical and instrumental evaluation. Plast Reconstr Surg Glob Open, 4, e1027, 2016.

43.

Vranckx

J.J.

Ex vivo gene transfer to full thickness wounds. A platform for autologous ‘smart’ tissue engineering for tissue repair (Thesis-report). Leuven, Belgium: Acco publishers, 2007. ISBN 978-9-082-28060-9

44.

Hendrickx

, Verdonck

, Van Den Berge

, et al. Integration of blood outgrowth endothelial cells in dermal fibroblast sheets promotes full thickness wound healing. Stem Cells, 28, 1165, 2010.

45.

Hoshi

, Fujihara

, Saijo

, et al. Implant-type Tissue-engineered cartilage for secondary correction of cleft lip-nose patients: an exploratory first-in-human trial. J Clin Trials, 07, 1, 2017.

46.

Hoshi

, Fujihara

, Saijo

, et al. Three-dimensional changes of noses after transplantation of implant-type tissue-engineered cartilage for secondary correction of cleft lip–nose patients. Regen Ther, 7, 72, 2017.

47.

Zhou

, Jiang

, Yin

, et al. In Vitro Regeneration of Patient-specific Ear-shaped cartilage and its first clinical application for auricular reconstruction. EBioMedicine, 28, 287, 2018.

48.

Yang

, Xiao

, Wang

, Li

, Kong

, and Liao

The immune reaction and degradation fate of scaffold in cartilage/bone tissue engineering. Mater Sci Eng C, 104, 109927, 2019.

49.

Jiang

, and Tuan

R.S.

Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol, 11, 206, 2015.

50.

Caldwell

K.L.

, and Wang

Cell-based articular cartilage repair: the link between development and regeneration. Osteoarthr Cartil, 23, 351, 2015.

51.

O'Sullivan

, D'Arcy

, Barry

F.P.

, Murphy

J.M.

, and Coleman

C.M.

Mesenchymal chondroprogenitor cell origin and therapeutic potential. Stem Cell Res Ther, 2, 8, 2011.

52.

Williams

, Khan

I.M.

, Richardson

, et al. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage. PLoS One, 5, e13246, 2010.

53.

Martin

, Vunjak-Novakovic

, Yang

, Langer

, and Freed

L.E.

Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue. Exp Cell Res, 253, 681, 1999.

54.

Pomerantseva

, Bichara

D.A.

, Tseng

, et al. Ear-shaped stable auricular cartilage engineered from extensively expanded chondrocytes in an immunocompetent experimental animal model. Tissue Eng Part A, 22, 197, 2016.

55.

Liu

, Li

, Yin

, et al. Prolonged in vitro precultivation alleviates post-implantation inflammation and promotes stable subcutaneous cartilage formation in a goat model. Biomed Mater, 12, 015006, 2016.

56.

Vanlauwe

, Saris

D.B.F.

, Victor

, Almqvist

K.F.

, Bellemans

, and Luyten

F.P.

Five-year outcome of characterized chondrocyte implantation versus microfracture for symptomatic cartilage defects of the knee: early treatment matters. Am J Sports Med, 39, 2566, 2011.

57.

Lenas

, Moos

, and Luyten

F.P.

Developmental engineering: a new paradigm for the design and manufacturing of cell-based products. Part II: from genes to networks: tissue engineering from the viewpoint of systems biology and network science. Tissue Eng Part B Rev, 15, 395, 2009.

58.

Bartlett

, Skinner

J.A.

, Gooding

C.R.

, et al. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee. A prospective, randomised study. J Bone Joint Surg B, 87, 640, 2005.

59.

Saris

, Price

, Widuchowski

, et al. Matrix-applied characterized autologous cultured chondrocytes versus microfracture. Am J Sports Med, 42, 1384, 2014.

60.

Mumme

, Barbero

, Miot

, et al. Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial. Lancet, 388, 1985, 2016.

61.

Atala

, Bauer

S.B.

, Soker

, Yoo

J.J.

, and Retik

A.B.

Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 367, 1241, 2006.

62.

Bhargava

, Patterson

J.M.

, Inman

R.D.

, MacNeil

, and Chapple

C.R.

Tissue-engineered buccal mucosa urethroplasty—clinical outcomes. Eur Urol, 53, 1263, 2008.

63.

Raya-Rivera

, Esquiliano

D.R.

, Yoo

J.J.

, Lopez-Bayghen

, Soker

, and Atala

Tissue-engineered autologous urethras for patients who need reconstruction: an observational study. Lancet, 377, 1175, 2011.

64.

Fossum

, Skikuniene

, Orrego

, and Nordenskjöld

Prepubertal follow-up after hypospadias repair with autologous in vitro cultured urothelial cells. Acta Paediatr, 101, 755, 2012.

65.

Engel

, Ram-Liebig

, Reiß

, et al. Tissue engineered buccal mucosa urethroplasty. Outcome of our first 10 patients. J Urol, 187, e6, 2012.

66.

Joseph

D.B.

, Borer

J.G.

, De Filippo

R.E.

, Hodges

S.J.

, and McLorie

G.A.

Autologous Cell Seeded Biodegradable Scaffold for Augmentation Cystoplasty: Phase II Study in Children and Adolescents with Spina Bifida. J Urol, 191, 1389, 2014.

67.

Bivalacqua

, Steinberg

, Smith

, et al. 178 Pre-clinical and clinical translation of a tissue engineered neo–urinary conduit using adipose derived smooth muscle cells for urinary reconstruction. Eur Urol Suppl, 13, e178, 2014.

68.

Ram-Liebig

, Barbagli

, Heidenreich

, et al. Results of use of tissue-engineered autologous oral mucosa graft for urethral reconstruction: a multicenter, prospective, observational trial. EBioMedicine, 23, 185, 2017.

69.

Barbagli

, Akbarov

, Heidenreich

, et al. Anterior urethroplasty using a new tissue engineered oral mucosa graft: surgical techniques and outcomes. J Urol, 200, 448, 2018.

70.

Shin'Oka

, Imai

, and Ikada

Transplantation of a tissue-engineered pulmonary artery [5]. N Engl J Med, 344, 532, 2001.

71.

Naito

, Imai

, Shin'oka

, et al. Successful clinical application of tissue-engineered graft for extracardiac Fontan operation. J Thorac Cardiovasc Surg, 125, 419, 2003.

72.

Shin'oka

, Matsumura

, Hibino

, et al. Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells. J Thorac Cardiovasc Surg, 129, 1330, 2005.

73.

McAllister

T.N.

, Maruszewski

, Garrido

S.A.

, et al. Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study. Lancet, 373, 1440, 2009.

74.

Wystrychowski

, Cierpka

, Zagalski

, et al. Case study: first implantation of a frozen, devitalized tissueengineered vascular graft for urgent hemodialysis access. J Vasc Access, 12, 67, 2011.

75.

Sugiura

, Matsumura

, Miyamoto

, Miyachi

, Breuer

C.K.

, and Shinoka

Tissue-engineered vascular grafts in children with congenital heart disease: intermediate term follow-up. Semin Thorac Cardiovasc Surg, 30, 175, 2018.

76.

Chachques

J.C.

, Trainini

J.C.

, Lago

, et al. Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium (MAGNUM Clinical Trial): One Year Follow-Up. Cell Transplant, 16, 927, 2007.

77.

Chachques

J.C.

, Trainini

J.C.

, Lago

, Cortes-Morichetti

, Schussler

, and Carpentier

Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium (MAGNUM Trial): Clinical Feasibility Study. Ann Thorac Surg, 85, 901, 2008.

78.

Olausson

, Patil

P.B.

, Kuna

V.K.

, et al. Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study. Lancet, 380, 230, 2012.

79.

Paolo

, Prato

, Rotundo

, et al. Case series an autologous cell hyaluronic acid graft technique for gingival augmentation: a case series. J Periodontol, 2, 262, 2003.

80.

Mohammadi

, Shokrgozar

M.A.

, and Mofid

Culture of Human Gingival Fibroblasts on a Biodegradable Scaffold and Evaluation of Its Effect on Attached Gingiva: A Randomized, Controlled Pilot Study. J Periodontol, 78, 1897, 2007.

81.

McGuire

M.K.

, and Nunn

M.E.

Evaluation of the Safety and Efficacy of Periodontal Applications of a Living Tissue-Engineered Human Fibroblast-Derived Dermal Substitute. I. Comparison to the Gingival Autograft: A Randomized Controlled Pilot Study. J Periodontol, 76, 867, 2005.

82.

Murata

, Okuda

, Momose

, Kubo

, Kuroyanagi

, and Wolff

L.F.

Root coverage with cultured gingival dermal substitute composed of gingival fibroblasts and matrix: a case series. Int J Periodontics Restorative Dent, 28, 461, 2008.

83.

Jhaveri

H.M.

, Chavan

M.S.

, Tomar

G.B.

, Deshmukh

V.L.

, Wani

M.R.

, and Miller

P.D.

Acellular Dermal Matrix Seeded With Autologous Gingival Fibroblasts for the Treatment of Gingival Recession: A Proof-of-Concept Study. J Periodontol, 81, 616, 2010.

84.

Peña

, Junquera

L.M.

, Llorente

, De Villalaín

, De Vicente

J.C.

, and Llames

Clinical outcomes after the use of complete autologous oral mucosa equivalents: preliminary cases. Oral Surg Oral Med Oral Pathol Oral Radiol, 113, e4, 2012.

85.

Llames

, Recuero

, Romance

, et al. Tissue-engineered oral mucosa for mucosal reconstruction in a pediatric patient with hemifacial microsomia and ankyloglossia. Cleft Palate-Craniofacial J, 51, 246, 2014.

86.

Milinkovic

, Aleksic

, Jankovic

, et al. Clinical application of autologous fibroblast cell culture in gingival recession treatment. J Periodontal Res, 50, 363, 2015.

87.

Sieira Gil

, Pagés

C.M.

, Díez

E.G.

, Llames

, Fuertes

A.F.

, and Vilagran

J.L.

Tissue-engineered oral mucosa grafts for intraoral lining reconstruction of the maxilla and mandible with a fibula flap. J Oral Maxillofac Surg, 73, 195.e1, 2015.

88.

Buda

, Vannini

, Cavallo

, Grigolo

, Cenacchi

, and Giannini

Osteochondral lesions of the knee: a new one-step repair technique with bone-marrow-derived cells. J Bone Joint Surg Am, 92(Suppl 2), 2, 2010.

89.

Wakitani

, Nawata

, Tensho

, Okabe

, Machida

, and Ohgushi

Repair of articular cartilage defects in the patello-femoral joint with autologous bone marrow mesenchymal cell transplantation: three case reports involving nine defects in five knees. J Tissue Eng Regen Med, 1, 74, 2007.

90.

Enea

, Cecconi

, Calcagno

, et al. Single-stage cartilage repair in the knee with microfracture covered with a resorbable polymer-based matrix and autologous bone marrow concentrate. Knee, 20, 562, 2013.

91.

Kang

, Liu

, Guan

, et al. Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage. Biomaterials, 33, 4535, 2012.

92.

Best

, Onwuka

, Pepper

, Sams

, Breuer

, and Breuer

Cardiovascular tissue engineering: preclinical validation to bedside application. Physiology (Bethesda), 31, 7, 2016.

93.

Matsumura

, Miyagawa-Tomita

, Shin'Oka

, Ikada

, and Kurosawa

First evidence that bone marrow cells contribute to the construction of tissue-engineered vascular autografts in vivo. Circulation, 108, 1729, 2003.

94.

Vranckx, J.J., Yao, F., and Eriksson, E. Gene Transfer of Growth Factors for Wound Repair. In: Rovee, Maibach

, H., eds. The Epidermis in Wound Healing. Boca Raton, FL: CRC Press, 2004, pp. 265–283.

95.

Madry

, Alini

, Stoddart

M.J.

, Evans

, Miclau

, and Steiner

Barriers and strategies for the clinical translation of advanced orthopaedic tissue engineering protocols. Eur Cells Mater, 27(Suppl), 17, 2014.

96.

O'Donnell

B.T.

, Ives

C.J.

, Mohiuddin

O.A.

, and Bunnell

B.A.

Beyond the present constraints that prevent a wide spread of tissue engineering and regenerative medicine approaches. Front Bioeng Biotechnol, 7, 95, 2019.

97.

Bertram

T.A.

, Tentoff

, Johnson

P.C.

, Tawil

, Van Dyke

, and Hellman

K.B.

Hurdles in tissue engineering/regenerative medicine product commercialization: a pilot survey of governmental funding agencies and the financial industry. Tissue Eng Part A, 18, 2187, 2012.

98.

Selmi

T.A.S.

, Verdonk

, Chambat

, et al. Autologous chondrocyte implantation in a novel alginate-agarose hydrogel. J Bone Joint Surg Br, 90-B, 597, 2008.

99.

Nehrer

, Chiari

, Domayer

, Barkay

, and Yayon

Results of Chondrocyte Implantation with a Fibrin-Hyaluronan Matrix: A Preliminary Study. Clin Orthop Relat Res, 466, 1849, 2008.

100.

Kon

, Gobbi

, Filardo

, Delcogliano

, Zaffagnini

, and Marcacci

Arthroscopic second-generation autologous chondrocyte implantation compared with microfracture for chondral lesions of the knee. Am J Sports Med, 37, 33, 2009.

101.

Zeifang

, Oberle

, Nierhoff

, Richter

, Moradi

, and Schmitt

Autologous chondrocyte implantation using the original periosteum-cover technique versus matrix-associated autologous chondrocyte implantation. Am J Sports Med, 38, 924, 2010.

102.

Schneider

, Rackwitz

, Andereya

, et al. A Prospective Multicenter Study on the Outcome of Type I Collagen Hydrogel–Based Autologous Chondrocyte Implantation (CaReS) for the Repair of Articular Cartilage Defects in the Knee. Am J Sports Med, 39, 2558, 2011.

103.

Clavé

, Potel

J.F.

, Servien

, Neyret

, Dubrana

, and Stindel

Third-generation autologous chondrocyte implantation versus mosaicplasty for knee cartilage injury: 2-year randomized trial. J Orthop Res, 34, 658, 2016.

104.

Zak

, Albrecht

, Wondrasch

, et al. Results 2 Years After Matrix-Associated Autologous Chondrocyte Transplantation Using the Novocart 3D Scaffold. Am J Sports Med, 42, 1618, 2014.

105.

Anderson

D.E.

, Williams

R.J.

, DeBerardino

T.M.

, et al. Magnetic resonance imaging characterization and clinical outcomes after neocart surgical therapy as a primary reparative treatment for knee cartilage injuries. Am J Sports Med, 45, 875, 2017.

106.

Chang

S.C.N.

, Tobias

, Roy

A.K.

, Vacanti

C.A.

, and Bonassar

L.J.

Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding. Plast Reconstr Surg, 112, 793, 2003.

107.

Rotter

, Ung

, Roy

A.K.

, et al. Role for Interleukin 1α in the Inhibition of chondrogenesis in autologous implants using polyglycolic acid–polylactic acid scaffolds. Tissue Eng, 11, 192, 2005.

108.

Morotomi

, Wada

, Uehara

, Enjo

, and Isogai

Effect of local environment, fibrin, and basic fibroblast growth factor incorporation on a canine autologous model of bioengineered cartilage tissue. Cells Tissues Organs, 196, 398, 2012.

109.

Ding

, Chen

B.O.

, Lv

, et al. Tissue engineering and regenerative medicine bone marrow mesenchymal stem cell-based engineered cartilage ameliorates polyglycolic acid/polylactic acid scaffold-induced inflammation through m2 polarization of macrophages in a pig model. Stem Cells Transl Med, 8, 1079, 2016.

110.

Clark

E.S.

, Best

, Onwuka

, et al. Effect of cell seeding on neotissue formation in a tissue engineered trachea. J Pediatr Surg, 51,:49, 2016.

111.

Pepper

V.K.

, Onwuka

E.A.

, Best

C.A.

, et al. Endoscopic management of tissue-engineered tracheal graft stenosis in an ovine model. Laryngoscope, 127, 2219, 2017.

112.

Gilevich

I.V.

, Sotnichenko

A.S.

, Karal-ogly

D.D.

, et al. In vivo experimental study of biological compatibility of tissue engineered tracheal construct in laboratory primates. Bull Exp Biol Med, 164, 770, 2018.