Physical, Mechanical, and Biological Properties of Fibrin Scaffolds for Cartilage Repair.

Articular cartilage is a highly organized tissue that provides remarkable load-bearing and low friction properties, allowing for smooth movement of diarthrodial joints; however, due to the avascular, aneural, and non-lymphatic characteristics of cartilage, joint cartilage has self-regeneration and repair limitations. Cartilage tissue engineering is a promising alternative for chondral defect repair. It proposes models that mimic natural tissue structure through the use of cells, scaffolds, and signaling factors to repair, replace, maintain, or improve the specific function of the tissue. In chondral tissue engineering, fibrin is a biocompatible biomaterial suitable for cell growth and differentiation with adequate properties to regenerate damaged cartilage. Additionally, its mechanical, biological, and physical properties can be enhanced by combining it with other materials or biological components. This review addresses the biological, physical, and mechanical properties of fibrin as a biomaterial for cartilage tissue engineering and as an element to enhance the regeneration or repair of chondral lesions.

Application of combined porous tantalum scaffolds loaded with bone morphogenetic protein 7 to repair of osteochondral defect in rabbits.

PURPOSE: Porous tantalum (PT) has been widely used in orthopaedic applications for low modulus of elasticity, excellent biocompatibility, and the microstructures similar to cancellous bone. In order to improve the biological activity of PT, biologically active factors can be combined with the material. The purpose of this study was to investigate if bone morphogenetic protein 7 (BMP-7) modifications could enhance the repairing of cartilage of PT in osteochondral defect in medial femoral condyle of rabbits. METHODS: A cylindrical osteochondral defect model was created on the animal medial femoral condyle of and filled as follows: PT modified with BMP-7 for MPT group, non-modified PT for the PT group, while no implants were used for the blank group. The regenerated osteochondral tissue was assessed and analyzed by histological observations at four, eight and 16 weeks post-operation and evaluated in an independent and blinded manner by five different observers using a histological score. Osteochondral and subchondral bone defect repair was assessed by micro-CT scan at 16 weeks post-operation, while the biomechanical test was performed at 16 weeks post-operation. RESULTS: Briefly, higher overall histological score was observed in the MPT group compared to PT group. Furthermore, more new osteochondral tissue and bone formed at the interface and inside the inner pores of scaffolds of the MPT group compared to PT group. Additionally, the micro-CT data suggested that the new bone volume fractions and the quantity and quality of trabecular bone, as well as the maximum release force of the bone, were higher in the MPT group compared to PT group. CONCLUSIONS: We demonstrated that the applied modified PT with BMP-7 promotes excellent subchondral bone regeneration and may serve as a novel approach for osteochondral defects repair.

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy.

Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-ß1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Hypoxia Differentially Affects Chondrogenic Differentiation of Progenitor Cells from Different Origins.

Background and Objectives: Ear cartilage malformations are commonly encountered problems in reconstructive surgery, since cartilage has low self-regenerating capacity. Malformations that impose psychological and social burden on one's life are currently treated using ear prosthesis, synthetic implants or autologous flaps from rib cartilage. These approaches are challenging because not only they request high surgical expertise, but also they lack flexibility and induce severe donor-site morbidity. Through the last decade, tissue engineering gained attention where it aims at regenerating human tissues or organs in order to restore normal functions. This technique consists of three main elements, cells, growth factors, and above all, a scaffold that supports cells and guides their behavior. Several studies have investigated different scaffolds prepared from both synthetic or natural materials and their effects on cellular differentiation and behavior. Methods and Results: In this study, we investigated a natural scaffold (alginate) as tridimensional hydrogel seeded with progenitors from different origins such as bone marrow, perichondrium and dental pulp. In contact with the scaffold, these cells remained viable and were able to differentiate into chondrocytes when cultured in vitro. Quantitative and qualitative results show the presence of different chondrogenic markers as well as elastic ones for the purpose of ear cartilage, upon different culture conditions. Conclusions: We confirmed that auricular perichondrial cells outperform other cells to produce chondrogenic tissue in normal oxygen levels and we report for the first time the effect of hypoxia on these cells. Our results provide updates for cartilage engineering for future clinical applications.

Increasing Collagen to Bioink Drives Mesenchymal Stromal Cells-Chondrogenesis from Hyaline to Calcified Layers.

The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.

Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy.

BACKGROUND: Articular cartilage repair using implantable photocrosslinkable hydrogels laden with chondrogenic cells, represents a promising in situ cartilage engineering approach for surgical treatment. The development of a surgical procedure requires a minimal viable product optimized for the clinical scenario. In our previous work we demonstrated how gelatin based photocrosslinkable hydrogels in combination with infrapatellar derived stem cells allow the production of neocartilage in vitro. In this study, we aim to optimize the critical facets of the in situ cartilage engineering therapy: the cell source, the cell isolation methodology, the cell expansion protocol, the cell number, and the delivery approach. METHODS: We evaluated the impact of the critical facets of the cell-laden hydrogel therapy in vitro to define an optimized protocol that was then used in a rabbit model of cartilage repair. We performed cells counting and immunophenotype analyses, chondrogenic potential evaluation via immunostaining and gene expression, extrusion test analysis of the photocrosslinkable hydrogel, and clinical assessment of cartilage repair using macroscopic and microscopic scores. RESULTS: We identified the adipose derived stem cells as the most chondrogenic cells source within the knee joint. We then devised a minimally manipulated stem cell isolation procedure that allows a chondrogenic population to be obtained in only 85 minutes. We found that cell expansion prior to chondrogenesis can be reduced to 5 days after the isolation procedure. We characterized that at least 5 million of cells/ml is needed in the photocrosslinkable hydrogel to successfully trigger the production of neocartilage. The maximum repairable defect was calculated based on the correlation between the number of cells retrievable with the rapid isolation followed by 5-day non-passaged expansion phase, and the minimum chondrogenic concentration in photocrosslinkable hydrogel. We next optimized the delivery parameters of the cell-laden hydrogel therapy. Finally, using the optimized procedure for in situ tissue engineering, we scored superior cartilage repair when compared to the gold standard microfracture approach. CONCLUSION: This study demonstrates the possibility to repair a critical size articular cartilage defect by means of a surgical streamlined procedure with optimized conditions.

Integration of Bioglass Into PHBV-Constructed Tissue-Engineered Cartilages to Improve Chondrogenic Properties of Cartilage Progenitor Cells.

Background: The Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffold has proven to be a promising three-dimensional (3D) biodegradable and bioactive scaffold for the growth and proliferation of cartilage progenitor cells (CPCs). The addition of Bioglass into PHBV was reported to increase the bioactivity and mechanical properties of the bioactive materials. Methods: In the current study, the influence of the addition of Bioglass into PHBV 3D porous scaffolds on the characteristics of CPC-based tissue-engineered cartilages in vivo were compared. CPCs were seeded into 3D macroporous PHBV scaffolds and PHBV/10% Bioglass scaffolds. The CPC-scaffold constructs underwent 6 weeks in vitro chondrogenic induction culture and were then transplanted in vivo for another 6 weeks to evaluate the difference between the CPC-PHBV construct and CPC-PHBV/10% Bioglass construct in vivo. Results: Compared with the pure PHBV scaffold, the PHBV/10% Bioglass scaffold has better hydrophilicity and a higher percentage of adhered cells. The CPC-PHBV/10%Bioglass construct produced much more cartilage-like tissues with higher cartilage-relative gene expression and cartilage matrix protein production and better biomechanical performance than the CPC-PHBV construct. Conclusion: The addition of Bioglass into 3D PHBV macroporous scaffolds improves the characteristics of CPC-based tissue-engineered cartilages in vivo.

A Collagen-Conducting Polymer Composite with Enhanced Chondrogenic Potential.

INTRODUCTION: Conducting polymers (CPs) have demonstrated promise for promoting tissue repair, yet their ability to facilitate cartilage regeneration has yet to be thoroughly investigated. Integrating CPs into common scaffolds for tissue regeneration, such as collagen, would enable mechanistic studies on the potential for CPs to promote cartilage repair. Here, we combine absorbable collagen sponges (ACS) with the CP PEDOT-S and show that the PEDOT-S-collagen composite (PEDOT-ACS) has enhanced chondrogenic potential compared to the collagen sponge alone. METHODS: PEDOT-S was incorporated through a simple incubation process. Changes to scaffold topography, elastic modulus, swelling ratio, and surface charge were measured to analyze how PEDOT-S affected the material properties of the scaffold. Changes in rat bone marrow mesenchymal stem cell (rBMSC) functionality were assessed with cell viability and glycosaminoglycan production assays. RESULTS: Macrostructure and microstructure of the scaffold remained largely unaffected by PEDOT-S modification, as observed through SEM images and quantification of scaffold porosity. Zeta potential, swelling ratio, and dry elastic modulus of the collagen scaffold were significantly changed by the incorporation of PEDOT-S. Seeding cells on PEDOT-ACS improved cell viability and enhanced glycosaminoglycan production. CONCLUSION: We demonstrate a practical approach to generate PEDOT-S composites with comparable physical properties to pristine collagen scaffolds. We show that PEDOT-ACS can influence cell functionality and serve as a promising model system for mechanistic investigations on the roles of bioelectronic signaling in the repair of cartilage and other tissue types.

3D Bioprinting Photo-Crosslinkable Hydrogels for Bone and Cartilage Repair.

Three-dimensional (3D) bioprinting has become a promising strategy for bone manufacturing, with excellent control over geometry and microarchitectures of the scaffolds. The bioprinting ink for bone and cartilage engineering has thus become the key to developing 3D constructs for bone and cartilage defect repair. Maintaining the balance of cellular viability, drugs or cytokines' function, and mechanical integrity is critical for constructing 3D bone and/or cartilage scaffolds. Photo-crosslinkable hydrogel is one of the most promising materials in tissue engineering; it can respond to light and induce structural or morphological transition. The biocompatibility, easy fabrication, as well as controllable mechanical and degradation properties of photo-crosslinkable hydrogel can meet various requirements of the bone and cartilage scaffolds, which enable it to serve as an effective bio-ink for 3D bioprinting. Here, in this review, we first introduce commonly used photo-crosslinkable hydrogel materials and additives (such as nanomaterials, functional cells, and drugs/cytokine), and then discuss the applications of the 3D bioprinted photo-crosslinkable hydrogel scaffolds for bone and cartilage engineering. Finally, we conclude the review with future perspectives about the development of 3D bioprinting photo-crosslinkable hydrogels in bone and cartilage engineering.

Oxygen-generating microparticles in chondrocytes-laden hydrogels by facile and versatile click chemistry strategy.

Currently, oxygen supply for in vitro cell culture is one of the major challenges in tissue engineering, especially in three-dimensional (3D) structures, such as polymeric hydrogels, because oxygen is an essential element for cells survival. In this context, oxygen levels must be maintained in articular cartilage to promote the differentiation, viability, and proliferation of chondrocytes due to the low level of oxygen presence in this region. Although some technologies employ oxygen-generating materials to add sufficient oxygen levels, the limitations and challenges of current technologies include the lack of controlled, sustained, and prolonged release of the oxygen. Moreover, the fabrication methods may leave some impurities or residues resulting in toxicity to the cells. "Click" chemistry is a facile, versatile, and compatible chemical strategy to engineer hydrogels for tissue engineering applications. Herein, we disclose the engineering of oxygen-generating microparticles in chondrocytes-laden hydrogels through a versatile catalyst-free tetrazine and norbornene inverse electron demand Diels‒Alder (iEDDA) click reaction. The hydrogels combine chondroitin sulfate (CS) and poly(ethylene glycol) (PEG) crosslinked in situ, displaying tunable rheological and mechanical properties, for sustained and prolonged oxygen-release. Gene expression analysis of the chondrocytes by real-time reverse transcription polymerase chain reaction (RT-PCR) demonstrated promising cell response within the engineered hydrogel.

Fabrication of a novel 3D scaffold for cartilage tissue repair: In-vitro and in-vivo study.

Self-repairing is not an advanced ability of articular cartilage. Tissue engineering has provided a novel way for reconstructing cartilage using natural polymers because of their biocompatibility and bio-functionality. The purpose of cartilage tissue engineering is to design a scaffold with proper pore structure and similar biological and mechanical properties to the native tissue. In this study, porous scaffolds prepared from gelatin, chitosan and silk fibroin were blended with varying ratios. Between the blends of chitosan (C), gelatin (G) and silk fibroin (S), the scaffold with the weight per volume ratio of 2:2:3 (w/v) showed the most favorable and higher certain properties than the other blends. The CGS 2:2:3 scaffold showed the best pore size that is between 100 µm and 300 µm. The water absorption and degradation rate of the CGS 2:2:3 scaffold were found suitable for cartilage tissue engineering. Cell culture study using human chondrocytes showed good cell adhesion and proliferation. To further study the effect of this scaffold on the living tissue, 36 rabbits were randomly assigned to CGS 2:2:3 scaffold with and without seeded chondrocytes and control groups. Hematoxylin and Eosin (H&E), Masson's trichrome (MT), and safranin O (SO) staining showed 65 ± 9.1% new cartilage tissue present in the defect filled with cell-seeded scaffold and most of the cartilaginous tissue was hyaline cartilage, while the control group showed no new cartilage tissue.

Engineering Cartilage Tissue by Co-culturing of Chondrocytes and Mesenchymal Stromal Cells.

Co-culture of chondrocytes and mesenchymal stromal cells (MSCs) has been shown to be beneficial in engineering cartilage tissue in vitro. In these co-cultures, MSCs increase the proliferation and matrix deposition of chondrocytes. The MSCs accomplish this beneficial effect by so-called trophic actions. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe different methods for making co-cultures of MSCs and chondrocytes. We also provide detailed protocols for analyzing MSC-chondrocyte co-cultures with cell tracking, proliferation assays, species-specific polymerase chain reactions (PCR), rheological analysis, compression analysis, RNA-sequencing analysis, short tandem repeats analysis, and biochemical examination.

3D-Printed Reinforcement Scaffolds with Targeted Biodegradation Properties for the Tissue Engineering of Articular Cartilage.

Achieving regeneration of articular cartilage is challenging due to the low healing capacity of the tissue. Appropriate selection of cell source, hydrogel, and scaffold materials are critical to obtain good integration and long-term stability of implants in native tissues. Specifically, biomechanical stability and in vivo integration can be improved if the rate of degradation of the scaffold material matches the stiffening of the sample by extracellular matrix secretion of the encapsulated cells. To this end, a novel 3D-printed lactide copolymer is presented as a reinforcement scaffold for an enzymatically crosslinked hyaluronic acid hydrogel. In this system, the biodegradable properties of the reinforced scaffold are matched to the matrix deposition of articular chondrocytes embedded in the hydrogel. The lactide reinforcement provides stability to the soft hydrogel in the early stages, allowing the composite to be directly implanted in vivo with no need for a preculture period. Compared to pure cellular hydrogels, maturation and matrix secretion remain unaffected by the reinforced scaffold. Furthermore, excellent biocompatibility and production of glycosaminoglycans and collagens are observed at all timepoints. Finally, in vivo subcutaneous implantation in nude mice shows cartilage-like tissue maturation, indicating the possibility for the use of these composite materials in one-step surgical procedures.

Electrospun Polymers in Cartilage Engineering-State of Play.

Articular cartilage defects remain a clinical challenge. Articular cartilage defects progress to osteoarthritis, which negatively (e.g., remarkable pain, decreased mobility, distress) affects millions of people worldwide and is associated with excessive healthcare costs. Surgical procedures and cell-based therapies have failed to deliver a functional therapy. To this end, tissue engineering therapies provide a promise to deliver a functional cartilage substitute. Among the various scaffold fabrication technologies available, electrospinning is continuously gaining pace, as it can produce nano- to micro- fibrous scaffolds that imitate architectural features of native extracellular matrix supramolecular assemblies and can deliver variable cell populations and bioactive molecules. Herein, we comprehensively review advancements and shortfalls of various electrospun scaffolds in cartilage engineering.

Stem Cells and Extrusion 3D Printing for Hyaline Cartilage Engineering.

Hyaline cartilage is deficient in self-healing properties. The early treatment of focal cartilage lesions is a public health challenge to prevent long-term degradation and the occurrence of osteoarthritis. Cartilage tissue engineering represents a promising alternative to the current insufficient surgical solutions. 3D printing is a thriving technology and offers new possibilities for personalized regenerative medicine. Extrusion-based processes permit the deposition of cell-seeded bioinks, in a layer-by-layer manner, allowing mimicry of the native zonal organization of hyaline cartilage. Mesenchymal stem cells (MSCs) are a promising cell source for cartilage tissue engineering. Originally isolated from bone marrow, they can now be derived from many different cell sources (e.g., synovium, dental pulp, Wharton's jelly). Their proliferation and differentiation potential are well characterized, and they possess good chondrogenic potential, making them appropriate candidates for cartilage reconstruction. This review summarizes the different sources, origins, and densities of MSCs used in extrusion-based bioprinting (EBB) processes, as alternatives to chondrocytes. The different bioink constituents and their advantages for producing substitutes mimicking healthy hyaline cartilage is also discussed.

A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model.

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.

Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering.

Osteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering.

Adult Dermal Stem Cells for Scaffold-Free Cartilage Tissue Engineering: Exploration of Strategies.

Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-ß1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-ß1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Respective stemness and chondrogenic potential of mesenchymal stem cells isolated from human bone marrow, synovial membrane, and synovial fluid.

BACKGROUND: MSCs isolated from bone marrow (BM-MSCs) have well-established chondrogenic potential, but MSCs derived from the synovial membrane (SM-MSCs) and synovial fluid (SF-MSCs) are thought to possess superior chondrogenicity. This study aimed to compare the in vitro immunophenotype and trilineage and chondrogenic potential of BM-MSCs to SM-MSCs and SF-MSCs. METHODS: MSCs were isolated from bone marrow (BM-MSCs), synovial membrane (SM-MSCs), and synovial fluid (SF-MSCs) extracted from the hips (BM) and knees (SM and SF) of advanced OA patients undergoing arthroplasty. Flow cytometric analysis was used at P2 to evaluate cell stemness. The trilinear differentiation test was performed at P2. At P3, MSC-seeded collagen sponges were cultured in chondrogenic medium for 28 days. Chondrogenic gene expression was quantified by qRT-PCR. Finally, the implants were stained to assess the deposition of proteoglycans and type II collagen. RESULTS: Despite variability, the immunophenotyping of BM-MSCs, SM-MSCs, and SF-MSCs was quite similar. All cell types were positive for the expression of stem cell markers and negative for exclusion markers. Additionally, chondrogenic differentiation and hypertrophy were more pronounced in BM-MSCs (ACAN, SOX9, COL2B, and COL10A) than in SF-MSCs, with SM-MSCs having intermediate characteristics. Concerning matrix synthesis, the three cell types were equipotent in terms of GAG content, while BM-MSC ECM synthesis of type II collagen was superior. CONCLUSIONS: Chondrogenic MSCs are easily collected from SM and SF in advanced human OA, but in vitro chondrogenesis that is superior to age-matched BM-MSCs should not be expected. However, due to intra-articular priming, SF-MSCs did not overexpress hypertrophic gene.