Cartilage-derived Matrix Research Articles

Improved Outcomes with Arthroscopic Bone Marrow Aspirate Concentrate and Cartilage-Derived Matrix Implantation versus Chondroplasty for the Treatment of Focal Chondral Defects of the Knee Joint: A Retrospective Case Series

PurposeTo compare the outcomes of patients undergoing treatment of focal chondral defects (FCDs) of the knee joint with chondroplasty versus bone marrow aspirate concentrate (BMAC) and cartilage-derived matrix (CDM) implantation.MethodsA retrospective chart review was performed for patients diagnosed with Outerbridge grade 3-4 FCDs. Patients were included if they were treated arthroscopically with BMAC/CDM implantation or chondroplasty alone between March 2016 and May 2019 and had more than 1-year follow-up. Postoperative outcomes included the visual analog scale (VAS) for pain; University of California, Los Angeles (UCLA) activity scores; Knee Outcome Survey (KOS) Activities of Daily Living (ADL) and Sports subscores; postoperative corticosteroid or hyaluronic acid injections; subsequent surgeries; and conversion to total knee arthroplasty.ResultsA total of 98 patients were identified with a mean follow-up in BMAC/CDM of 24 months (range 13-41 months) and in chondroplasty of 44 months (range 34-55 months). A subanalysis was performed to control for significant differences in age, which yielded 39 patients, ages 40-60 years. Within the subanalysis group, mean VAS scores were significantly lower in the BMAC/CDM group (1.7 vs 4.4; P = .005) and mean UCLA scores were significantly greater (7.1 vs 5.0; P = .002). Mean improvement in VAS and UCLA scores were similar between the BMAC/CDM and chondroplasty groups (–3.7 vs –1.3; P = .71, 1.9 vs 0.1; P = .14, respectively). Mean KOS ADL and Sports subscores were significantly greater among patients in the BMAC/CDM group (87% vs 55%; P = .001, 71% vs 41%; P = .002, respectively). There were no differences in postoperative injections, subsequent surgeries, or conversion to total knee arthroplasty between the BMAC/CDM and chondroplasty groups.ConclusionsPatients with grade 3-4 FCDs of the knee had improved postoperative outcomes when treated with BMAC/CDM implantation versus chondroplasty alone, as evidenced by a significant improvement in VAS and UCLA scores and significantly greater postoperative KOS ADL, and KOS Sport subscores.Level of EvidenceIV, therapeutic case series.

The use of a novel deer antler decellularized cartilage-derived matrix scaffold for repair of osteochondral defects

BackgroundThe physiologic regenerative capacity of cartilage is severely limited. Current studies on the repair of osteochondral defects (OCDs) have mainly focused on the regeneration of cartilage tissues. The antler cartilage is a unique regenerative cartilage that has the potential for cartilage repair.MethodsAntler decellularized cartilage-derived matrix scaffolds (adCDMs) were prepared by combining freezing-thawing and enzymatic degradation. Their DNA, glycosaminoglycans (GAGs), and collagen content were then detected. Biosafety and biocompatibility were evaluated by pyrogen detection, hemolysis analysis, cytotoxicity evaluation, and subcutaneous implantation experiments. adCDMs were implanted into rabbit articular cartilage defects for 2 months to evaluate their therapeutic effects.ResultsAdCDMs were observed to be rich in collagen and GAGs and devoid of cells. AdCDMs were also determined to have good biosafety and biocompatibility. Both four- and eight-week treatments of OCDs showed a flat and smooth surface of the healing cartilage at the adCDMs filled site. The international cartilage repair society scores (ICRS) of adCDMs were significantly higher than those of controls (porcine dCDMs and normal saline) (p < 0.05). The repaired tissue in the adCDM group was fibrotic with high collagen, specifically, type II collagen.ConclusionsWe concluded that adCDMs could achieve excellent cartilage regeneration repair in a rabbit knee OCDs model. Our study stresses the importance and benefits of adCDMs in bone formation and overall anatomical reconstitution, and it provides a novel source for developing cartilage-regenerating repair materials.

Improved Short-Term Outcomes of Osteochondral Lesions of the Knee Following Arthroscopic Treatment With Bone Marrow Aspirate Concentrate and Cartilage-Derived Matrix

PurposeTo assess the postoperative objective, subjective, and functional outcomes as well as complication rates in osteochondral defect patients treated with bone marrow aspirate concentrate (BMAC) and cartilage-derived matrix (CDM) during knee arthroscopy.MethodsA retrospective chart review was performed for patients treated arthroscopically with BMAC and CDM between August 2015 and August 2018 and had more than 1-year follow-up. Demographic factors such as age, sex, body mass index, and comorbidities were collected for all patients. Size and location of the osteochondral lesions also were documented.ResultsA total of 14 patients were identified with a mean follow-up of 19 months. On average, patients were 34 years of age (range 16-58 years) and 43% were female. Postoperatively, knee flexion increased by 8° from 124° to 132° (P = .002). All patients regained full extension; however, 1 patient later acquired a 2° extension contracture after a traumatic event. The average hamstring strength significantly increased from 4.1 to 4.6 postoperatively (P = .33). The average quadriceps strength significantly increased from 4.0 to 4.5 postoperatively (P = .007). Mean visual analog scale scores significantly decreased postoperatively (4.5 vs 1.4; P = .001). There was a significant increase in Knee Outcome Survey Activities of Daily Living scores (53.8 vs 92.9; P = .007). Mean Knee Outcome Survey-Sports scores also increased, although this was nonsignificant (28.2 vs 79.5; P = .560). No significant differences were noted in pain and functional outcomes when stratified by the osteochondral defect size and location. Complications included a stitch abscess, Baker’s cyst, and residual pain treated with hyaluronic acid injection.ConclusionsThis study demonstrated arthroscopic BMAC and CDM implantation appears to be safe and has the potential to improve patient outcomes in the short-term postoperative period.Level of EvidenceIV, therapeutic case series.

Extracellular Matrix Scaffold Using Decellularized Cartilage for Hyaline Cartilage Regeneration.

The repair of osteochondral defects is among the top ten medical needs of humans in the 21st centuries with many countries facing rapidly aging population involved with osteoarthritis as a major contributor to global disease burden. Tissue engineering methods have offered new windows of hope to treat such disorders and disabilities. Regenerative approaches to cartilage injuries require careful replication of the complex microenvironment of the native tissue. The decellularized hyaline cartilage derived from human allografts or xenografts is potentially an ideal scaffold, simulating the mechanical and biochemical properties, as well as biological microarchitecture of the hyaline cartilage. There have been many attempts to regenerate clinically viable hyaline cartilage tissue using decellularized cartilage-derived extracellular matrix with stem cell technology. This chapter describes the reproducible methods for hyaline cartilage decellularization and recellularization. In addition, quality control and characterization requirements of the product at each step, as well as the clinical applications of final product have been discussed.

Cartilage Particles can Promote Chondrogenesis of Adipose-Derived Stromal Cells on Poly(ε-Caprolactone)/Fibrin Hybrid Constructs Prepared via Sandwich Model

Electrospun fibers have demonstrated a remarkable potential as a framework structure in the fabrication of cartilage tissue engineering (CTE) scaffolds. Various extracellular matrices have been incorporated into electrospun scaffolds to mimic and simulate the extracellular environment. The objective of this study was to fabricate hybrid constructs using composite electrospun scaffolds based on poly (ε-caprolactone) (PCL) and cartilage-derived matrix (CDM) and fibrin hydrogel to improve the viability and differentiation of human adipose-derived stromal cells (ADSCs) for CTE applications.Initially, PCL and PCL-CDM electrospun mats were fabricated. Fibrin/ ADSCs hydrogel were seeded on PCL- CDM mats and arranged layer-by-layer using sandwich technique. This method has been employed to increase cell seeding and infiltration efficiency through the entire mass of the scaffold. Real-time reverse-transcription polymerase chain reaction (RT- PCR), were performed to examine the expression of collagen types II and X, SOX9 and aggrecan. The production of glycosaminoglycan (GAG) was also tested in vitro by Toluidine blue stain and biochemical assay in the cultured scaffolds.The findings demonstrated that incorporation of CDM in PCL fibers results in improved cell viability. Hematoxylin and eosin staining showed that the sandwich method resulted in homogenous cell seeding within the scaffold. Overall, the RT- PCR, biochemical and histological results, showed that incorporation of the CDM into PCL/fibrin sandwich scaffolds stimulated ADSCs chondrogenesis and produced the products which increased expression of chondrogenic genes. It also, enhanced GAG synthesis compared to PCL/fibrin scaffolds.These findings suggest PCL-CDM/fibrin can be considered as an appropriate hybrid scaffold for CTE applications.

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.

Inhibition of nucleotide pyrophosphatase/phosphodiesterase 1: implications for developing a calcium pyrophosphate deposition disease modifying drug.

Calcium pyrophosphate deposition (CPPD) is associated with osteoarthritis and is the cause of a common inflammatory articular disease. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) is the major ecto-pyrophosphatase in chondrocytes and cartilage-derived matrix vesicles (MVs). Thus, eNPP1 is a principle contributor to extracellular pyrophosphate levels and a potential target for interventions aimed at preventing CPPD. Recently, we synthesized and described a novel eNPP1-specific inhibitor, SK4A, and we set out to evaluate whether this inhibitor attenuates nucleotide pyrophosphatase activity in human OA cartilage. Cartilage tissue, chondrocytes and cartilage-derived MVs were obtained from donors with OA undergoing arthroplasty. The effect of SK4A on cell viability was assayed by the XTT method. eNPP1 expression was evaluated by western blot. Nucleotide pyrophosphatase activity was measured by a colorimetric assay and by HPLC analysis of adenosine triphosphate (ATP) levels. ATP-induced calcium deposition in cultured chondrocytes was visualized and quantified with Alizarin red S staining. OA chondrocytes expressed eNPP1 in early passages, but this expression was subsequently lost upon further passaging. Similarly, significant nucleotide pyrophosphatase activity was only detected in early-passage chondrocytes. The eNPP1 inhibitor, SK4A, was not toxic to chondrocytes and stable in culture medium and human plasma. SK4A effectively inhibited nucleotide pyrophosphatase activity in whole cartilage tissue, in chondrocytes and in cartilage-derived MVs and reduced ATP-induced CPPD. Nucleotide analogues such as SK4A may be developed as potent and specific inhibitors of eNPP1 for the purpose of lowering extracellular pyrophosphate levels in human cartilage with the aim of preventing and treating CPPD disease.

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability.

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model

To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaPscaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the+P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P<0.0001). The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the-P group integrated well with the recipient bone. The study stresses the importance of long-term invivo studies to assess the efficacy of cartilage repair techniques.

502. Biomaterial-Mediated Lentiviral Delivery of Anti-Inflammatory Genes in Cartilage-Derived Matrix Hemispheres

Objectives: Cartilage tissue engineering seeks to provide a functional replacement for damaged or degenerated articular surfaces that develop in osteoarthritis (OA). Human bone marrow-derived mesenchymal stem cells (MSCs) seeded within cartilage-derived matrix (CDM) hemispherical scaffolds exhibit robust chondrogenic differentiation and extracellular matrix (ECM) deposition without evidence of a hypertrophic phenotype. However, inflammation within an OA joint may inhibit chondrogenesis of MSCs and induce degradation of both native and engineered cartilage, particularly through the action of interleukin-1 (IL-1). The goal of this study was to develop an anatomically-shaped, tissue-engineered cartilage capable of tunable and inducible expression of IL-1 receptor antagonist (IL-1Ra) in MSCs via biomaterial-mediated lentiviral gene delivery. Methods: IL-1Ra or eGFP coding sequences were cloned into doxycycline(dox)-inducible lentiviral vectors. Hemispherical scaffolds were fabricated from resuspended cartilage powder in hemispherical molds (outer radius 4.76 mm, inner radius 3.175 mm). Scaffolds were crosslinked and sterilized via dehydrothermal treatment. Dox-inducible eGFP or IL-1Ra lentivirus was immobilized to PLL-coated CDM scaffolds to transduce MSCs upon seeding. Non-transduced (NT), eGFP-expressing, or IL-1Ra-expressing constructs were treated with 0 or 0.1 ng/mL IL-1 during 28 days of chondrogenesis (+10 ng/mL TGF-s3) with 1 µg/mL dox. Results: Transduction of MSCs occurred throughout the CDM hemispheres (Fig 1AFig 1A) and the transduction efficiency of MSCs isolated from the construct was 57±5% eGFP+ by flow cytometry. Hemispherical constructs maintained their anatomical shape throughout chondrogenic culture (Fig 1BFig 1B). IL-1Ra-expressing constructs produced over 200 ng/mL of IL-1Ra, a concentration expected to inhibit pathologic IL-1 concentrations, and IL-1 treatment did not affect IL-1Ra production (Fig 1CFig 1C). IL-1 treatment reduced ECM deposition by NT and eGFP-expressing MSCs but not IL-1Ra-expressing MSCs, as evidenced by safranin-O/fast green staining for glycosaminoglycans and total collagen, respectively (Fig 1DFig 1D). After 28 days of chondrogenesis, IL-1 treatment significantly increased matrix metalloproteinase (MMP) activity in the conditioned media of NT and eGFP-expressing constructs but not in IL-1Ra-expressing constructs (p<0.05) (Fig 1EFig 1E). Significance: We engineered an anatomically and compositionally mimetic cartilage hemisphere that is inflammation-resistant and shows promise for total articular resurfacing in an arthritic environment.Tunable production of IL-1Ra from an IL-1-resistant cartilage tissue may provide therapeutic effects to other joint tissues, such as the synovium or subchondral bone.View Large Image | Download PowerPoint Slide

Fabrication of anatomically-shaped cartilage constructs using decellularized cartilage-derived matrix scaffolds

The native extracellular matrix of cartilage contains entrapped growth factors as well as tissue-specific epitopes for cell-matrix interactions, which make it a potentially attractive biomaterial for cartilage tissue engineering. A limitation to this approach is that the native cartilage extracellular matrix possesses a pore size of only a few nanometers, which inhibits cellular infiltration. Efforts to increase the pore size of cartilage-derived matrix (CDM) scaffolds dramatically attenuate their mechanical properties, which makes them susceptible to cell-mediated contraction. In previous studies, we have demonstrated that collagen crosslinking techniques are capable of preventing cell-mediated contraction in CDM disks. In the current study, we investigated the effects of CDM concentration and pore architecture on the ability of CDM scaffolds to resist cell-mediated contraction. Increasing CDM concentration significantly increased scaffold mechanical properties, which played an important role in preventing contraction, and only the highest CDM concentration (11% w/w) was able to retain the original scaffold dimensions. However, the increase in CDM concentration led to a concomitant decrease in porosity and pore size. Generating a temperature gradient during the freezing process resulted in unidirectional freezing, which aligned the formation of ice crystals during the freezing process and in turn produced aligned pores in CDM scaffolds. These aligned pores increased the pore size of CDM scaffolds at all CDM concentrations, and greatly facilitated infiltration by mesenchymal stem cells (MSCs). These methods were used to fabricate of anatomically-relevant CDM hemispheres. CDM hemispheres with aligned pores supported uniform MSC infiltration and matrix deposition. Furthermore, these CDM hemispheres retained their original architecture and did not contract, warp, curl, or splay throughout the entire 28-day culture period. These findings demonstrate that given the appropriate fabrication parameters, CDM scaffolds are capable of maintaining complex structures that support MSC chondrogenesis.

In vivo degradation profile of porcine cartilage-derived extracellular matrix powder scaffolds using a non-invasive fluorescence imaging method

We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Cartilage-derived extracellular matrix extract promotes chondrocytic phenotype in three-dimensional tissue culture

Cell transplantation is a promising regenerative therapy for cartilage degeneration. However, obtaining sufficient numbers of cells for this purpose is a challenge, due a lack of autologous donor tissue and the difficulty of culturing chondrocytes in vitro. Tissue engineering strategies that induce or maintain chondrocytic phenotype may solve these problems by (1) broadening the range of available donor tissue, and (2) facilitating the expansion of these cells while controlling phenotypic drift. In this study, bone marrow-derived mesenchymal stem cells (MSCs) and cartilage-derived cells (CDCs) were cultured on composite hydrogels containing agarose and homogenized cartilage extracellular matrix (ECM). MSCs cultured on agarose-ECM scaffolds did not show significant signs of chondrogenic differentiation in the absence of additional cues. However, CDCs cultured on agarose-ECM scaffolds proliferated more rapidly than their ECM-free counterparts and MSCs, while retaining chondrocytic morphology. These results were corroborated via expression of cartilage marker genes: in autologous constructs, SOX 9 expression was upregulated by 12.6 ± 5.3-fold, and COL II was upregulated by 2.0 ± 0.3-fold. Agarose-ECM composite hydrogels are therefore useful for expanding partially differentiated CDCs for applications in regenerative medicine.

Decellularized Cartilage-Derived Matrix as Substrate for Endochondral Bone Regeneration

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles

The natural process of endochondral bone formation in the growing skeletal system is increasingly inspiring the field of bone tissue engineering. However, in order to create relevant-size bone grafts, a cell carrier is required that ensures a high diffusion rate and facilitates matrix formation, balanced by its degradation. Therefore, we set out to engineer endochondral bone in gelatin methacrylamide (GelMA) hydrogels with embedded multipotent stromal cells (MSCs) and cartilage-derived matrix (CDM) particles. CDM particles were found to stimulate the formation of a cartilage template by MSCs in the GelMA hydrogel in vitro. In a subcutaneous rat model, this template was subsequently remodeled into mineralized bone tissue, including bone-marrow cavities. The GelMA was almost fully degraded during this process. There was no significant difference in the degree of calcification in GelMA with or without CDM particles: 42.5 ± 2.5% vs. 39.5 ± 8.3% (mean ± standard deviation), respectively. Interestingly, in an osteochondral setting, the presence of chondrocytes in one half of the constructs fully impeded bone formation in the other half by MSCs. This work offers a new avenue for the engineering of relevant-size bone grafts, by the formation of endochondral bone within a degradable hydrogel.

Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.

ObjectiveAlthough extracellular matrix (ECM)–derived scaffolds have been extensively studied and applied in a number of clinical applications, the use of ECM as a biomaterial for (osteo)chondral regeneration is less extensively explored. This study aimed at evaluating the chondrogenic potential of cells seeded on cartilage-derived matrix (CDM) scaffolds in vitro.DesignScaffolds were generated from decellularized equine articular cartilage and seeded with either chondrocytes or multipotent stromal cells (MSCs). After 2, 4, and 6 weeks of in vitro culture, CDM constructs were analyzed both histologically (hematoxylin and eosin, Safranin-O, collagen types I and II) and biochemically (glycosaminoglycan [GAG] and DNA content).ResultsAfter 4 weeks, both cell types demonstrated chondrogenic differentiation; however, the MSCs significantly outperformed chondrocytes in producing new GAG-containing cartilaginous matrix.ConclusionThese promising in vitro results underscore the potency of CDM scaffolds in (osteo)chondral defect repair.

Electrospun cartilage-derived matrix scaffolds for cartilage tissue engineering

Macroscale scaffolds created from cartilage-derived matrix (CDM) demonstrate chondroinductive or chondro-inductive properties, but many fabrication methods do not allow for control of nanoscale architecture. In this regard, electrospun scaffolds have shown significant promise for cartilage tissue engineering. However, nanofibrous materials generally exhibit a relatively small pore size and require techniques such as multilayering or the inclusion of sacrificial fibers to enhance cellular infiltration. The objectives of this study were (1) to compare multilayer to single-layer electrospun poly(ɛ-caprolactone) (PCL) scaffolds for cartilage tissue engineering, and (2) to determine whether incorporation of CDM into the PCL fibers would enhance chondrogenesis by human adipose-derived stem cells (hASCs). PCL and PCL-CDM scaffolds were prepared by sequential collection of 60 electrospun layers from the surface of a grounded saline bath into a single scaffold, or by continuous electrospinning onto the surface of a grounded saline bath and harvest as a single-layer scaffold. Scaffolds were seeded with hASCs and evaluated over 28 days in culture. The predominant effects on hASCs of incorporation of CDM into scaffolds were to stimulate sulfated glycosaminoglycan synthesis and COL10A1 gene expression. Compared with single-layer scaffolds, multilayer scaffolds enhanced cell infiltration and ACAN gene expression. However, compared with single-layer constructs, multilayer PCL constructs had a much lower elastic modulus, and PCL-CDM constructs had an elastic modulus approximately 1% that of PCL constructs. These data suggest that multilayer electrospun constructs enhance homogeneous cell seeding, and that the inclusion of CDM stimulates chondrogenesis-related bioactivity.

The effects of crosslinking of scaffolds engineered from cartilage ECM on the chondrogenic differentiation of MSCs

Scaffolds fabricated from cartilage extracellular matrix provide a chondroinductive environment that stimulates cartilaginous matrix synthesis in a variety of cell types. A limitation of these cartilage-derived matrix (CDM) scaffolds is that they contract during in vitro culture, which unpredictably alters their shape. The current study examined the hypothesis that collagen crosslinking techniques could inhibit cell-mediated contraction of CDM scaffolds. We analyzed the effects of dehydrothermal (DHT) treatment, ultraviolet light irradiation (UV), and the chemical crosslinker carbodiimide (CAR) on scaffold contraction and chondrogenic differentiation of adult human bone marrow-derived stem cells (MSCs). Both physical and chemical crosslinking treatments retained the original scaffold dimensions. DHT and UV treatments produced significantly higher glycosaminoglycan and collagen contents than CAR crosslinked and non-crosslinked constructs. Crosslinking treatments influenced the composition of newly synthesized matrix, and DHT treatment best matched the composition of native cartilage. DHT, UV, and non-crosslinked CDM films supported cell attachment, while CAR crosslinking inhibited cell adhesion. These results affirm that collagen crosslinking treatments can prevent cell-mediated contraction of CDM scaffolds. Interestingly, crosslinking treatments influence chondrogenic differentiation. These effects seem to be mediated by modifications to cell-matrix interactions between MSCs and the CDM; however, further work is necessary to elucidate the specific mechanisms involved in this process.