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Background: Current clinical approaches to regenerate temporomandibular joint (TMJ) articulating cartilage defects only treat the symptoms (i.e. pain and dysfunction) and do not seek to restore joint integrity for long-term relief. Therefore, we investigated a novel self-assembling tissue-engineered cartilage to overcome this significant clinical issue for TMJ regenerative purposes., Objectives: Examine the maturation of dynamic self-regenerating cartilage (dSRC) using auricular chondrocytes and evaluate a novel combinatorial approach with fractional laser treatment and dSRC implantation for TMJ cartilage repair., Materials and Methods: A suspension of 10 7 freshly harvested rabbit ear chondrocytes was cultured under a continuous reciprocating motion to form the dSRC. After 2, 4 and 8 weeks of culture, dSRC samples were stained with H&E, Safranin-O and Toluidine Blue. Immunohistochemistry (IHC) was performed for collagens type I and II. Channels (300-500 μm diameter and 1.2-1.5 mm depth) were created in six freshly harvested condyles using a fractional Erbium laser. Two groups were tested: dSRC in a laser-ablated lesion (experimental) and an empty laser-ablated channel (control). TMJ condyles were cultured for up to 8 weeks and analysed as described above., Results: H&E staining showed a high cell density in dSRC compared to native cartilage. All dSRC groups demonstrated intense Safranin-O staining, indicating high glycosaminoglycan (GAG) production and intense Toluidine Blue staining showed high proteoglycan content. IHC confirmed that dSRC consisted predominantly of collagen type II. The experimental group showed improved cartilage repair at both time points compared to the empty channels., Conclusion: dSRC viability and successful matrix formation were demonstrated in vitro. The combination of fractional laser ablation and dSRC implantation enhanced cartilage repair., (© 2024 John Wiley & Sons Ltd.)

Successful 3D scaffold designs for musculoskeletal tissue engineering necessitate full consideration of the form and function of the tissues of interest. When designing structures for engineering cartilage and osteochondral tissues, one must reconcile the need to develop a mechanically robust system that maintains the health of cells embedded in the scaffold. In this work, we present an approach that decouples the mechanical and biochemical needs and allows for the independent development of the structural and cellular niches in a scaffold. Using the highly tuned capabilities of digital light processing-based stereolithography, structures with complex architectures are achieved over a range of effective porosities and moduli. The 3D printed structure is infilled with mesenchymal stem cells and soft biomimetic hydrogels, which are specifically formulated with extracellular matrix analogs and tethered growth factors to provide selected biochemical cues for the guided differentiation towards chondrogenesis and osteogenesis. We demonstrate the ability to utilize these structures to (a) infill a focal chondral defect and mitigate macroscopic and cellular level changes in the cartilage surrounding the defect, and (b) support the development of a stratified multi-tissue scaffold for osteochondral tissue engineering., (© 2021 IOP Publishing Ltd.)

Cartilage tissue engineering strategies often rely on hydrogels with fixed covalent crosslinks for chondrocyte encapsulation, yet the resulting material properties are largely elastic and can impede matrix deposition. To address this limitation, hydrazone crosslinked poly(ethylene glycol) hydrogels were formulated to achieve tunable viscoelastic properties and to study how chondrocyte proliferation and matrix deposition vary with the time-dependent material properties of covalent adaptable networks. Hydrazone equilibrium differences were leveraged to produce average stress relaxation times from hours (4.01 × 10 3  s) to months (2.78 × 10 6  s) by varying the percentage of alkyl-hydrazone (aHz) and benzyl-hydrazone (bHz) crosslinks. Swelling behavior and degradation associated with adaptability were characterized to quantify temporal network changes that can influence the behavior of encapsulated chondrocytes. After four weeks, mass swelling ratios varied from 36 ± 3 to 17 ± 0.4 and polymer retention ranged from 46 ± 4% to 92 ± 5%, with higher aHz content leading to loss of network connectivity with time. Hydrogels were formulated near the Flory-Stockmayer bHz percolation threshold (17% bHz) to investigate chondrocyte response to distinct levels of covalent architecture adaptability. Four weeks post-encapsulation, formulations with average relaxation times of 3 days (2.6 × 10 5 s) revealed increased cellularity and an interconnected articular cartilage-specific matrix. Chondrocytes embedded in this adaptable formulation (22% bHz) deposited 190 ± 30% more collagen and 140 ± 20% more sulfated glycosaminoglycans compared to the 100% bHz control, which constrained matrix deposition to pericellular space. Collectively, these findings indicate that incorporating highly adaptable aHz crosslinks enhanced regenerative outcomes. However, connected networks containing more stable bHz bonds were required to achieve the highest quality neocartilaginous tissue. STATEMENT OF SIGNIFICANCE: Covalently crosslinked hydrogels provide robust mechanical support for cartilage tissue engineering applications in articulating joints. However, these materials traditionally demonstrate purely elastic responses to deformation despite the dynamic viscoelastic properties of native cartilage tissue. Here, we present hydrazone poly(ethylene glycol) hydrogels with tunable viscoelastic properties and study covalent adaptable networks for cartilage tissue engineering. Using hydrazone equilibrium and Flory-Stockmayer theory we identified average relaxation times leading to enhanced regenerative outcomes and showed that extracellular matrix deposition was biphasic as a function of the hydrazone covalent adaptability. We also showed that the incorporation of highly adaptable covalent crosslinks could improve cellularity of neotissue, but that a percolating network of more stable bonds was required to maintain scaffold integrity and form the highest quality neocartilaginous tissue., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage maturation and prevented shrinkage and distortion. This is the first demonstration of a stable, ear-shaped elastic cartilage engineered from auricular chondrocytes that underwent clinical-scale expansion in an immunocompetent animal over an extended period of time.

The objective of this study was to assess the ability of tissue engineered cartilage to adhere to and integrate with porous polyethylene (PPE) in vivo and to evaluate the biomechanical integrity of the bond formed at the interface. Porcine auricular, articular, and costal chondrocytes were suspended in fibrin gel polymer and placed between discs of PPE to form tri-layer constructs. Controls consisted of fibroblasts suspended in gel or gel alone between the discs. Constructs were implanted into nude mice for 6, 12, and 18 weeks. Upon harvest, specimens were evaluated for neocartilage formation and integration into the PPE, using histological, dimensional (mass, thickness, diameter), and biomechanical (adhesion strength, interfacial stiffness, failure energy and failure strain) analyses. Neotissue was formed in all experimental constructs, consisting mostly of neocartilage integrating with discs of PPE. Control samples contained only fibrous tissue. Biomechanical analyses demonstrated that adhesion strength, interfacial stiffness, and failure energy were all significantly higher in the chondrocyte-seeded samples than in fibroblast-seeded controls, with the exception of costal constructs at 12 weeks, which were not significantly greater than controls. In general, failure strains did not vary between groups. In conclusion, porous polyethylene supported the growth of neocartilage that formed mechanically functional bonds with the PPE., (© 2014 Wiley Periodicals, Inc.)

Healing articular cartilage remains a significant clinical challenge because of its limited self-healing capacity. While delivery of autologous chondrocytes to cartilage defects has received growing interest, combining cell-based therapies with scaffolds that capture aspects of native tissue and promote cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold does not match the rate of production by cells leading to generally low extracellular matrix outputs. Here, a poly (ethylene glycol) (PEG) norbornene hydrogel is functionalized with thiolated transforming growth factor (TGF-β1) and cross-linked by an MMP-degradable peptide. Chondrocytes are co-encapsulated with a smaller population of mesenchymal stem cells, with the goal of stimulating matrix production and increasing bulk mechanical properties of the scaffold. The co-encapsulated cells cleave the MMP-degradable target sequence more readily than either cell population alone. Relative to non-degradable gels, cellularly degraded materials show significantly increased glycosaminoglycan and collagen deposition over just 14 d of culture, while maintaining high levels of viability and producing a more widely-distributed matrix. These results indicate the potential of an enzymatically degradable, peptide-functionalized PEG hydrogel to locally influence and promote cartilage matrix production over a short period. Scaffolds that permit cell-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Carved autologous costal cartilage and porous polyethylene implants (Medpor) are the most common approaches for total ear reconstruction, but these approaches may have inconsistent cosmetic outcomes, a high risk of extrusion, or other surgical complications. Engineering ear cartilage to emulate native auricular tissue is an appealing approach, but often the cell-seeded scaffolds are susceptible to shrinkage and architectural changes when placed in vivo. The aim of this study was to assess the most favorable conditions for in vitro pre-culture of cell-seeded type I collagen scaffolds prior to in vivo implantation., Methods: Sheep auricular chondrocytes were seeded into this type I collagen scaffold. The cell-seeded constructs were cultured in either static or dynamic conditions for two days or two weeks and then implanted into nude mice for another six weeks. The harvested constructs were evaluated histologically, immunohistochemically, and biochemically., Results: Robust neo-cartilage formation was found in these collagen scaffolds seeded with auricular chondrocytes, which was comparable to native cartilage morphologically, histologically, and biochemically. Culture under dynamic conditions prior to implantation improved the neo-cartilage formation histologically and biochemically., Conclusion: Dynamic culture of this cell-seeded fibrous collagen material could permit predictable engineered auricular cartilage and a promising approach for external ear reconstruction., (Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Objectives: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model., Methods: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infra-auricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented., Results: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks., Conclusions: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs.

Tissue-engineered cartilage has historically been an attractive alternative treatment option for auricular reconstruction. However, the ability to reliably generate autologous auricular neocartilage in an immunocompetent preclinical model should first be established. The objectives of this study were to demonstrate engineered autologous auricular cartilage in the immunologically aggressive subcutaneous environment of an immunocompetent animal model, and to determine the impact of in vitro culture duration of chondrocyte-seeded constructs on the quality of neocartilage maturation in vivo. Auricular cartilage was harvested from eight adult sheep; chondrocytes were isolated, expanded in vitro, and seeded onto fibrous collagen scaffolds. Constructs were cultured in vitro for 2, 6, and 12 weeks, and then implanted autologously in sheep and in control nude mice for 6 and 12 weeks. Explanted tissue was stained with hematoxylin and eosin, safranin O, toluidine blue, collagen type II, and elastin. DNA and glycosaminoglycans (GAGs) were quantified. The quality of cartilage engineered in sheep decreased with prolonged in vitro culture time. Superior cartilage formation was demonstrated after 2 weeks of in vitro culture; the neocartilage quality improved with increased implantation time. In nude mice, neocartilage resembled native sheep auricular cartilage regardless of the in vitro culture length, with the exception of elastin expression. The DNA quantification was similar in all engineered and native cartilage (p>0.1). All cartilage engineered in sheep had significantly less GAG than native cartilage (p<0.02); significantly more GAG was observed with increased implantation time (p<0.02). In mice, the GAG content was similar to that of native cartilage and became significantly higher with increased in vitro or in vivo durations (p<0.02). Autologous auricular cartilage was successfully engineered in the subcutaneous environment of an ovine model using expanded chondrocytes seeded on a fibrous collagen scaffold after a 2-week in vitro culture period.

Engineered cartilage is a promising option for auricular reconstruction. We have previously demonstrated that a titanium wire framework within a composite collagen ear-shaped scaffold helped to maintain the gross dimensions of the engineered ear after implantation, resisting the deformation forces encountered during neocartilage maturation and wound healing. The ear geometry was redesigned to achieve a more accurate aesthetic result when implanted subcutaneously in a nude rat model. A non-invasive method was developed to assess size and shape changes of the engineered ear in three dimensions. Computer models of the titanium framework were obtained from CT scans before and after implantation. Several parameters were measured including the overall length, width and depth, the minimum intrahelical distance and overall curvature values for each beam section within the framework. Local curvature values were measured to gain understanding of the bending forces experienced by the framework structure in situ. Length and width changed by less than 2%, whereas the depth decreased by approximately 8% and the minimum intrahelical distance changed by approximately 12%. Overall curvature changes identified regions most susceptible to deformation. Eighty-nine per cent of local curvature measurements experienced a bending moment less than 50 µN-m owing to deformation forces during implantation. These quantitative shape analysis results have identified opportunities to improve shape fidelity of engineered ear constructs.

Numerous studies on engineering cartilage utilizing chondrocytes from juvenile animal sources have been reported. However, there are many unknown aspects of engineering cartilage using human chondrocytes-especially from middle-aged or elderly adults-which are critical for clinical application of tissue engineering in the field of orthopedic surgery. The primary aim of this study was to engineer neocartilage tissue from 50-60-year-old human chondrocytes in comparison to engineered cartilage made from juvenile swine chondrocytes (JSCs). Articular chondrocytes from middle-aged, nonarthritic humans and juvenile swine were isolated and placed in culture for expansion. The chondrocytes (passage 1) were mixed in fibrin gel at 40-60×10(6) cells/mL until polymerization. Cells/nodule constructs and devitalized cartilage-cells/hydrogel-devitalized cartilage constructs (three-layered model) were implanted into subcutaneous pockets of nude mice for 12, 18, and 24 weeks. The specimens were evaluated histologically, biochemically, and biomechanically. This allowed for direct comparison of the cartilage engineered from human versus swine cells. Histological analysis demonstrated that samples engineered utilizing chondrocytes from middle-aged adults accumulated basophilic, sulfated glycosaminoglycans (sGAG), and abundant type II collagen around the cells in a manner similar to that seen in samples engineered using JSCs at all time points. Biochemical analysis revealed that samples made with human cells had about 40%-60% of the amount hydroxyproline of native human cartilage, a trend parallel to that observed in the specimens made with swine chondrocytes. The amount of sGAG in the human chondrocyte specimens was about one-and-a-half times the amount in native human cartilage, whereas the amount in the samples made with swine chondrocytes was always less than native cartilage. The biomechanical analysis revealed that the stiffness and tensile of samples made with human cells were in a pattern similar to that seen with swine chondrocytes. This study demonstrates that chondrogenesis using articular chondrocytes from middle-aged adults can be achieved in a predictable and reliable manner similar to that shown in studies using cells from juvenile animals and can form the basis of engineering cartilage with degradable scaffolds in this patient population.

The reconstruction, repair, and regeneration of the external auricular framework continue to be one of the greatest challenges in the field of tissue engineering. To replace like with like, we should emulate the native structure and composition of auricular cartilage by combining a suitable chondrogenic cell source with an appropriate scaffold under optimal in vitro and in vivo conditions. Due to the fact that a suitable and reliable substitute for auricular cartilage has yet to be engineered, hand-carved autologous costal cartilage grafts and ear-shaped porous polyethylene implants are the current treatment modalities for auricular reconstruction. However, over the last decade, significant advances have been made in the field of regenerative medicine and tissue engineering. A variety of scaffolds and innovative approaches have been investigated as alternatives to using autologous carved costal cartilage or porous polyethylene implants. A review of recent developments and the current state of the art and science is presented, focusing on scaffolds, cell sources, seeding densities, and mechanical characteristics of tissue-engineered auricular cartilage.

Engineered cartilage composed of a patient's own cells can become a feasible option for auricular reconstruction. However, distortion and shrinkage of ear-shaped constructs during scaffold degradation and neocartilage maturation in vivo have hindered the field. Scaffolds made of synthetic polymers often generate degradation products that cause an inflammatory reaction and negatively affect neocartilage formation in vivo. Porous collagen, a natural material, is a promising candidate; however, it cannot withstand the contractile forces exerted by skin and surrounding tissue during normal wound healing. We hypothesised that a permanent support in the form of a coiled wire embedded into a porous collagen scaffold will maintain the construct's size and ear-specific shape. Half-sized human adult ear-shaped fibrous collagen scaffolds with and without embedded coiled titanium wire were seeded with sheep auricular chondrocytes, cultured in vitro for up to 2 weeks, and implanted subcutaneously on the backs of nude mice. After 6 weeks, the dimensional changes in all implants with wire support were minimal (2.0% in length and 4.1% in width), whereas significant reduction in size occurred in the constructs without embedded wire (14.4% in length and 16.5% in width). No gross distortion occurred over the in vivo study period. There were no adverse effects on neocartilage formation from the embedded wire. Histologically, mature neocartilage extracellular matrix was observed throughout all implants. The amount of DNA, glycosaminoglycan, and hydroxyproline in the engineered cartilage were similar to that of native sheep ear cartilage. The embedded wire support was essential for avoiding shrinkage of the ear-shaped porous collagen constructs.

Purpose: Chondrocytes lose their ability to produce cartilaginous matrix during multiplication in culture through repeated passages, resulting in inferior tissue phenotype. To overcome the limited amount of primary chondrocytes, we aimed to determine the optimal culture condition for in vitro/in vivo cartilage regeneration using human adipose-derived mesenchymal stem cells (AMSCs)., Methods: To evaluate the effects exerted by the chondrocytic culture condition on AMSC, we utilized chondrocyte conditioned medium (CM) and/or co-culture methods to prime and differentiate AMSCs. We evaluated ultimate in vivo engineered cartilage with primed AMSCs with that of chondrocytes. To examine the link between conditioned factors and proliferation/differentiation, cell cycle progression of AMSCs were examined using 5-ethynyl-2'-deoxyuridine (EdU), and gene expression was monitored., Results: We report that AMSCs can be stimulated to become chondrogenic cells when expanded with chondrocyte CM. Polymeric scaffolds co-seeded with CM- expanded AMSCs and primary chondrocytes resulted in in vivo cartilaginous tissues with similar biochemical content to constructs seeded with chondrocytes alone., Conclusion: These results indicate that chondrocyte CM consists of suitable morphogenetic factors that induce the chondrogenic priming of AMSCs for cartilage tissue engineering.

The objective of this study was to fabricate hydrogel matrix-engineered biosynthetic cartilage using a porous poly(vinyl alcohol) hydrogel (PVA-H) and articular chondrocytes. Chondrocytes were suspended in fibrin gel (FG) or saline carriers and injected into porous PVA-H discs and three-layered constructs (PVA-H between devitalized cartilage). After implantation in nude mice, PVA discs were explanted at 6 weeks and subjected to creep testing for a 20 h period. The three-layered constructs were explanted at 12 weeks and subjected to tensile testing to determine the strength of the interface between the engineered hydrogel and devitalized cartilage. Histological analysis revealed PVA-H porous channels occupied by chondrocytes. Extracellular matrix was identified by Safranin-O and toluidine blue stains. Immunohistochemical analysis revealed a positive stain for COL II and scant staining for COL I. Creep and relaxation response of PVA-FG-chondrocyte constructs was similar to that of native cartilage. The presence of cells and FG significantly enhanced the integration strength of layered constructs (p < 0.05). These results demonstrate that porous PVA-H in combination with FG and chondrocytes provides a favorable microenvironment for tissue engineering of articular cartilage, creating a biosynthetic construct that can adhere to native devitalized articular cartilage utilizing hydrogel matrix-engineered technology.

In this study we investigated the histological, biochemical, and integrative features of the neocartilage using swine auricular chondrocytes photoencapsulated into two poly(ethylene glycol) dimethacrylate (PEGDM) copolymer hydrogels of a different degradation profile: degradable (PEG-4,5LA-DM) and nondegradable (PEGDM) macromers in molar ratios of 60:40 and 70:30. Integration of the engineered tissue with existing native cartilage was examined using an articular cartilaginous ring model. Experimental group samples (total n=96) were implanted subcutaneously into nude mice and harvested at 6, 12, and 18 weeks. Nonimplanted constructs (total n=16) were used as controls for quantification of DNA, glycosaminoglycan, and hydroxyproline. Histologically, neocartilage resembled both the cellular population and composition of the extracellular matrix of the native swine auricular cartilage. DNA content demonstrated that the photoencapsulated chondrocytes were capable of survival and proliferation over time. Both glycosaminoglycan and hydroxyproline contents appeared higher in the neotissue, which was supported by less degradable PEGDM hydrogel. Integration of neocartilage with surrounding native cartilage improved with time, resulting in the development of tight integration interface. PEGDM copolymer hydrogels can support in vivo chondrogenesis by photoencapsulating auricular chondrocytes.

Background: Limited options exist for the restoration of craniofacial cartilage. Autologous tissue or porous polyethylene is currently used for nasal and auricular reconstruction. Both options are associated with drawbacks, including donor site morbidity and implant extrusion. Poly(vinyl alcohol) (PVA) is a non-degradable flexible biocompatible polymer than can be engineered to mimic the properties of cartilage. The goal of this study was to engineer a biosynthetic hybrid construct using a combination of PVA-alginate hydrogels and human nasal septum chondrocytes., Materials and Methods: Chondrocytes isolated from human nasal septum cartilage were expanded and mixed with 2% sodium alginate hydrogel. The chondrocyte-alginate mix was injected into a non-degradable porous PVA hydrogel, creating biosynthetic constructs. A group of these constructs were implanted into the subcutaneous environment of nude mice, while the other group was cultured in a spinner flask bioreactor system for 10 d and then implanted. After 6 wk in vivo, the histologic, biochemical, and biomechanical properties were examined., Results: Histological analysis demonstrated sulfated glycosaminoglycans and deposition of collagen type II in constructs from both groups. Constructs cultured in the bioreactor system prior in vivo implantation demonstrated higher levels of DNA, glycosaminoglycans, and hydroxyproline. An increase of 22% in the compressive strength of the engineered constructs exposed to the bioreactor was also observed., Conclusion: A novel porous PVA-alginate gel hybrid was used to successfully engineer human cartilage in vivo. A 10-d period of bioreactor culturing increased levels of DNA, glycosaminoglycans, hydroxyproline, and the compressive modulus of the constructs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

This study's purpose was to investigate whether photochemically crosslinking collagen gel to encapsulate chondrocytes (articular, auricular, costal) would permit new cartilage formation in vivo, and to determine whether this neocartilage had the ability to integrate with existing native cartilage. Chondrocytes from swine were embedded in collagen gel that was photochemically crosslinked using riboflavin and visible light. Controls were collagen gels containing cells that were not crosslinked. Cylindrical implants (0.1 cc) were placed in athymic mice for 4 and 8 weeks. To study integration, the constructs were crosslinked within articular cartilage rings and implanted in the mice. Samples were analyzed in terms of macroscopic, microscopic, and biochemical aspects. Photocrosslinking did not affect the amount of glycosaminoglycan and type II collagen produced by the cells. We found that photochemical crosslinking collagen gel enhances the physical parameters of the gel and permits new cartilage formation that can integrate with existing native cartilage.

Collagen gels have many favorable attributes for tissue engineering, but the gels undergo dramatic contraction when cells are added because of the weak noncovalent bonds that form during spontaneous gelation. We hypothesized that photochemically cross-linking collagen gels would make suitable scaffolds for tissue engineering with favorable cell viability and minimal gel contraction. Rose Bengal and riboflavin were chosen as candidate photo-initiators for gel cross-linking using 532- and 458-nm-light wavelengths, respectively. Chondrocyte viability was measured after initial gelation for several concentrations of initiators. Cell viability and gel contraction were then measured using chondrocytes and fibroblasts over 7 days of culture. Rose Bengal used at concentrations necessary for gelation resulted in little or no cell viability. Short-term viability results showed that 0.25- or 0.5-mM concentrations of riboflavin, and 40 s of illumination permitted more than 90% cell viability. Using riboflavin concentrations of 0.25 or 0.5 mM, long-term chondrocyte viability was 113.1 +/- 11.6% and 25.4 +/- 2.7%, respectively, at day 7. Although non-cross-linked chondrocyte constructs contracted to 59.9 +/- 11.8% of their original diameter and fibroblasts contracted to 24.9 +/- 5.0% of their original diameter by day 7, the cross-linked constructs retained 88.8 +/- 7.4% and 85.5 +/- 5.0% of the original diameter, respectively. In conclusion, by photochemically cross-linking collagen gels using riboflavin and visible light, stable gel scaffolds with favorable cell survival can be produced.

Background: Meniscal tears in the avascular zones do not heal. Although tissue-engineering approaches using cells seeded onto scaffolds could expand the indication for meniscal repair, harvesting autologous cells could cause additional trauma to the patient. Allogenic cells, however, could provide an unlimited amount of cells., Hypothesis: Allogenic cells from 2 anatomical sources can repair lesions in the avascular region of the meniscus., Study Design: Controlled laboratory study., Methods: Both autologous and allogenic chondrocytes were seeded onto a Vicryl mesh scaffold and sutured into a bucket-handle lesion created in the medial menisci of 17 swine. Controls consisted of 3 swine knees treated with unseeded implants and controls from a previous experiment in which 4 swine were treated with suture only and 4 with no treatment. Menisci were harvested after 12 weeks and evaluated histologically for new tissue and percentage of interface healing surface; they were also evaluated statistically., Results: The lesions were closed in 15 of 17 menisci. None of the control samples demonstrated healing. Histologic analysis of sequential cuts through the lesion showed formation of new scar-like tissue in all experimental samples. One of 8 menisci was completely healed in the allogenic group and 2 of 9 in the autologous group; the remaining samples were partially healed in both groups. No statistically significant differences in the percentage of healing were observed between the autologous and allogenic cell-based implants., Conclusion: Use of autologous and allogenic chondrocytes delivered via a biodegradable mesh enhanced healing of avascular meniscal lesions., Clinical Relevance: This study demonstrates the potential of a tissue-engineered cellular repair of the meniscus using autologous and allogenic chondrocytes.

More than a decade of work has been devoted to engineering cartilage for articular surface repair. This review covers the use of fibrin gel polymer as an injectable scaffold for generating new cartilage matrix from isolated articular chondrocytes beginning with studies in mice and culminating in an applied study in swine joints. These studies began with developing a formulation of fibrin that was injectable and promoted cartilage matrix formation. Subsequent studies addressed the problems of volume loss after the scaffolds were placed in vivo by adding lyophilized cartilage matrix. Additional studies focused on the ability of isolated chondrocytes to heal and repair cartilage in a model that could be biomechanically tested. In conclusion, this series of studies demonstrated that fibrin gel is a suitable polymer gel for generating new cartilage matrix from articular chondrocytes. The new matrix is capable of forming mechanical bonds between cartilage disks and can lead to healing and integration. Armed with these results, implantation of fibrin-cell constructs into defects in swine knees showed new cartilage formation and filling of the defects. Continuing work in these models with fibrin and other polymerizable hydrogels could result in a suitable cell-based therapy for articular cartilage lesions.

This study investigated the in vivo formation of engineering cartilage within living or devitalized cartilage discs using reflectance mode confocal microscopy and conventional light microscopy. Pig articular chondrocytes were suspended in fibrin glue and placed between two cartilage discs. Four experimental groups were prepared: in groups 1 and 2, the cell-hydrogel composite was placed between two live or between two devitalized cartilage discs, respectively; in groups 3 and 4, acellular fibrin glue was placed between two live or between two devitalized cartilage discs, respectively. Samples were implanted in the back of nude mice and analyzed after 2, 5, and 8 weeks. Results showed that engineered cartilage seems to grow more homogenously when the cell-seeded gel was placed between devitalized cartilages than when it was placed between live cartilage matrices. Confocal microscopy provides valuable information on the integration of tissue-engineered cartilage with native tissue and could be useful for nondestructive imaging in vivo.

Background: Both native and engineered cartilage is brittle and fractures easily without perichondrium. The aim of this study was to understand the role of the perichondrium and try to enhance the flexible properties of tissue-engineered cartilage using expanded polytetrafluoroethylene (ePTFE) membrane as a pseudoperichondrium., Methods: The study was conducted in two phases. In phase I, native swine auricular cartilage of different thicknesses was studied by histologic evaluation and failure testing. Next, isolated perichondrium was bonded to native cartilage slices using fibrin glue or Dermabond and tested to failure. In phase II, swine auricular chondrocytes were suspended in fibrin glue. The chondrocyte-fibrin glue composites were then bound to expanded polytetrafluoroethylene membrane in two trilaminar configurations: In group EC-1, the membrane was in the center, whereas it was on the surfaces in group EC-2. Specimens were implanted into nude mice for 4 weeks, 8 weeks, 12 weeks, and 8 months and subjected to histologic evaluation and failure testing., Results: In phase I, the results demonstrated that perichondrium securely bonded to the cartilage plays an important role in maintaining the flexible nature of elastic cartilage. In phase II, failure testing revealed that specimens in group EC-1 (expanded polytetrafluoroethylene core) were fractured during bending and destroyed after torsion, whereas those in group EC-2 (cartilage core) returned to their original shape without fracturing even after rigorous torsion. Histologic analysis demonstrated that transplanted chondrocytes penetrated into the microporous structure of expanded polytetrafluoroethylene and created a bond to it., Conclusion: It is possible to engineer flexible cartilage using expanded polytetrafluoroethylene as a pseudoperichondrium.

Background: Previous attempts to engineer human ear-shaped constructs mimicked human shape but lacked the flexibility and size of a human ear. Recently, the authors engineered flexible cartilage by incorporating a perichondrium-like layer into the construct. In this study, they used lyophilized swine perichondrium as a pseudoperichondrium, examined its ability to confer flexibility to tissue-engineered cartilage, and used it to engineer flexible cartilage in the shape and size of a human ear., Methods: Auricular chondrocytes and perichondrium were isolated from swine. Chondrocytes were mixed with fibrin polymer and gelled to form 5 x 20-mm constructs. Constructs alone (control, n = 6) or constructs sandwiched between two layers of lyophilized swine perichondrium (experimental, n = 6) were implanted into athymic mice. Auricular chondrocytes in fibrin polymer and lyophilized perichondrium were also used to form a tri-layer, ear-shaped construct, which was implanted into an athymic rat and externally stented for 6 weeks (n = 1). At 12 weeks, constructs were analyzed with histology and gross mechanical testing., Results: New cartilaginous tissue was engineered in both the experimental and control groups. In samples laminated with lyophilized swine perichondrium, the intimate integration of the laminate with the neocartilage closely resembled the histoarchitecture of the native swine ear. Experimental constructs had mechanical properties similar to those of the native swine ear, while control constructs fractured with similar testing. The engineered ear could not be fractured with gross mechanical testing, and its size, shape, and flexibility remained stable., Conclusions: This study demonstrates that it is possible to engineer a cartilage construct that resembles the human ear not only in shape but also in size and flexibility. This study also confirms that lamination is a reliable method to confer elastic-like flexibility to an engineered cartilage construct.

Meniscus injury is common in today's active society. Despite the frequent presentation of meniscus injury, the decision to repair or resect a torn meniscus is not always straightforward. Current repair techniques are effective in the peripheral vascularized meniscus, but their success is not dependable in the avascularized zone. Tissue engineering, a discipline that combines the technologies of cell culture and biodegradable scaffolds to deliver a cellular repair, may be one future answer to this problem. The concept of using cell-based repair for torn menisci could improve healing of lesions in the avascular zone and broadly expand the indication for repair rather than removal, obviating the need for meniscectomy. This article reviews current advances in the relatively new field of tissue engineering toward the development of a tissue-engineered meniscal repair technique.

Articular chondrocytes can synthesize new cartilaginous matrix in vivo that forms functional bonds with native cartilage. Other sources of chondrocytes may have a similar ability to form new cartilage with healing capacity. This study evaluates the ability of various chondrocyte sources to produce new cartilaginous matrix in vivo and to form functional bonds with native cartilage. Disks of articular cartilage and articular, auricular, and costal chondrocytes were harvested from swine. Articular, auricular, or costal chondrocytes suspended in fibrin glue (experimental), or fibrin glue alone (control), were placed between disks of articular cartilage, forming trilayer constructs, and implanted subcutaneously into nude mice for 6 and 12 weeks. Specimens were evaluated for neocartilage production and integration into native cartilage with histological and biomechanical analysis. New matrix was formed in all experimental samples, consisting mostly of neocartilage integrating with the cartilage disks. Control samples developed fibrous tissue without evidence of neocartilage. Ultimate tensile strength values for experimental samples were significantly increased (p < 0.05) from 6 to 12 weeks, and at 12 weeks they were significantly greater (p < 0.05) than those of controls. We conclude that articular, auricular, and costal chondrocytes have a similar ability to produce new cartilaginous matrix in vivo that forms mechanically functional bonds with native cartilage.

Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.

A large-deflection elasticity model was used to describe the mechanical behavior of cartilaginous tissues during three-point bending tests. Force-deflection curves were measured for 20-mm long x 4-mm wide x approximately 1-mm thick strips of porcine auricular and costal cartilage. Using a least-squares method with elastic modulus in bending as the only adjustable parameter, data were fit to a model based on the von Karman theory for large deflection of plates. This model described the data well, with an average RMS error of 14.8% and an average R(2) value of 0.98. Using this method, the bending modulus of auricular cartilage (4.6 MPa) was found to be statistically lower (p < 0.05) than that of costal cartilage (7.1 MPa). Material features of the cartilage samples influenced the mechanical behavior, including the orientation of the perichondrium in auricular cartilage. These methods also were used to determine the elastic moduli of engineered cartilage samples produced by seeding chondrocytes into fibrin glue. The modulus of tissue-engineered constructs increased statistically with time (p < 0.05), but still were statistically lower than the moduli of the native tissue samples (p > 0.05), reaching only about a third of the values of native samples., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 597-602, 2004)

The primary goal of engineering cartilage as a therapeutic approach is to restore the physiological conditions of an affected or defective tissue in the body. Cartilage tissue is distributed widely in the human body and possesses an organization related to the specific demand of a particular anatomical region. In selecting the proper material for engineering cartilage, the functional demands of the replacement tissue must be considered. In summary, there is a multitude of scaffolds, naturally occurring and synthetic, that are suitable for engineering cartilage. Investigators have shown that the characteristics of the neocartilage differ significantly depending upon which scaffold is used. There are also large differences when a single scaffold is tested in vitro as opposed to in vivo. Moreover, the addition of other materials internally or externally to the cartilage composite influences the physical and biomechanical properties of the newly formed tissue. The results achieved so far are extremely encouraging and motivate further investigative efforts in the field. The biochemical composition and, more importantly, the biomechanical properties of the native tissue still represent the ideal replacement tissue.

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps. [ABSTRACT FROM AUTHOR]

Background: In the last decade, tissue‐engineering strategies for regenerating the temporomandibular joint (TMJ) have been investigated. This may be a promising strategy for the minimally invasive restoration of joint integrity. Objectives: To evaluate whether dental pulp stem cells (DPSCs) loaded in a light‐occured hydrogel made of gelatin methacryloyl (GelMA) enhance the regeneration of osteochondral defects in the rabbit TMJ. Materials and Methods: Defects were filled with GelMA alone (control group; n = 4) or filled with GelMA loaded with rabbit DPSCs (experimental group; n = 4), In one group, the TMJ capsule was opened without creating a defect (sham group; n = 2). The following micro‐CT parameters were analysed: bone volume to total volume ratio (BV/TV%) and bone mineral density (BMD). Histological evaluation was performed to assess cartilage regeneration features. A semi‐quantitative scoring system was also used to evaluate the defects. Results: All groups had no statistical difference regarding the micro‐CT parameters. The highest mean healing score was found for the experimental group. After 4 weeks, there were no signs of hydrogel in either group or no signs of inflammation in the adjacent tissues. The tissue formed in the defect was dense fibrous connective tissue. Conclusion: Adding DPSCs to GelMA did not provide a regenerative enhancement in TMJ osteochondral defects. This resulted in similar micro‐CT parameters after 4 weeks of healing, with improved signs of subchondral bone regeneration but no cartilage regeneration. [ABSTRACT FROM AUTHOR]

Autonomization is a physiological process allowing a flap to develop neo-vascularization from the reconstructed wound bed. This phenomenon has been used since the early application of flap surgeries but still remains poorly understood. Reconstructive strategies have greatly evolved since, and fasciocutaneous flaps have progressively replaced muscle-based reconstructions, ensuring better functional outcomes with great reliability. However, plastic surgeons still encounter challenges in complex cases where conventional flap reconstruction reaches its limitations. Furthermore, emerging bioengineering applications, such as decellularized scaffolds allowing a complex extracellular matrix to be repopulated with autologous cells, also face the complexity of revascularization. The objective of this article is to gather evidence of autonomization phenomena. A systematic review of flap autonomization is then performed to document the minimum delay allowing this process. Finally, past and potential applications in bio- and tissue-engineering approaches are discussed, highlighting the potential for in vivo revascularization of acellular scaffolds. [ABSTRACT FROM AUTHOR]

Osteoarthrosis is a debilitating disease affecting millions, yet engineering materials for cartilage regeneration has proven difficult because of the complex microstructure of this tissue. Articular cartilage, like many biological tissues, produces a time-dependent response to mechanical load that is critical to cell’s physiological function in part due to solid and fluid phase interactions and property variations across multiple length scales. Recreating the time-dependent strain and fluid flow may be critical for successfully engineering replacement tissues but thus far has largely been neglected. Here, microindentation is used to accomplish three objectives: (1) quantify a material’s time-dependent mechanical response, (2) map material properties at a cellular relevant length scale throughout zonal articular cartilage and (3) elucidate the underlying viscoelastic, poroelastic, and nonlinear poroelastic causes of deformation in articular cartilage. Untreated and trypsin-treated cartilage was sectioned perpendicular to the articular surface and indentation was used to evaluate properties throughout zonal cartilage on the cut surface. The experimental results demonstrated that within all cartilage zones, the mechanical response was well represented by a model assuming nonlinear biphasic behavior and did not follow conventional viscoelastic or linear poroelastic models. Additionally, 10% (w/w) agarose was tested and, as anticipated, behaved as a linear poroelastic material. The approach outlined here provides a method, applicable to many tissues and biomaterials, which reveals and quantifies the underlying causes of time-dependent deformation, elucidates key aspects of material structure and function, and that can be used to provide important inputs for computational models and targets for tissue engineering. Statement of Significance Elucidating the time-dependent mechanical behavior of cartilage, and other biological materials, is critical to adequately recapitulate native mechanosensory cues for cells. We used microindentation to map the time-dependent properties of untreated and trypsin treated cartilage throughout each cartilage zone. Unlike conventional approaches that combine viscoelastic and poroelastic behaviors into a single framework, we deconvoluted the mechanical response into separate contributions to time-dependent behavior. Poroelastic effects in all cartilage zones dominated the time-dependent behavior of articular cartilage, and a model that incorporates tension–compression nonlinearity best represented cartilage mechanical behavior. These results can be used to assess the success of regeneration and repair approaches, as design targets for tissue engineering, and for development of accurate computational models. [ABSTRACT FROM AUTHOR]

Background: The avascular portion of the meniscus cartilage in the knee does not have the ability to repair spontaneously. Hypothesis: Cell-based therapy is able to repair a lesion in the swine meniscus. Study Design: Controlled laboratory study. Methods: Sixteen Yorkshire pigs were divided into four groups. A longitudinal tear was produced in the avascular portion of the left medial meniscus of 4 pigs. Autologous chondrocytes were seeded onto devitalized allogenic meniscal slices and were secured inside the lesion with two sutures. Identical incisions were created in 12 other pigs, which were used as three separate control groups: 4 animals treated with an unseeded scaffold, 4 were simply sutured, and 4 were left untreated. Meniscal samples were collected after 9 weeks, and the samples were analyzed grossly, histologically, and histomorphometrically. Results: Gross results showed bonding of the lesion margins in the specimens of the experimental group, whereas no repair was noted in any of the control group specimens. Histological and histomorphometrical analysis showed multiple areas of healing in the specimens of the experimental group. Conclusions: This study demonstrated the ability of seeded chondrocytes to heal a meniscal tear. Clinical Relevance: Cell-based therapy could be a potential tool for avascular meniscus repair. [ABSTRACT FROM AUTHOR]