Decellularized Cartilage Extracellular Matrix Research Articles

Promotion of osteochondral repair through immune microenvironment regulation and activation of endogenous chondrogenesis via the release of apoptotic vesicles from donor MSCs

Utilizing transplanted human umbilical cord mesenchymal stem cells (HUMSCs) for cartilage defects yielded advanced tissue regeneration, but the underlying mechanism remain elucidated. Early after HUMSCs delivery to the defects, we observed substantial apoptosis. The released apoptotic vesicles (apoVs) of HUMSCs promoted cartilage regeneration by alleviating the chondro-immune microenvironment. ApoVs triggered M2 polarization in macrophages while simultaneously facilitating the chondrogenic differentiation of endogenous MSCs. Mechanistically, in macrophages, miR-100-5p delivered by apoVs activated the MAPK/ERK signaling pathway to promote M2 polarization. In MSCs, let-7i-5p delivered by apoVs promoted chondrogenic differentiation by targeting the eEF2K/p38 MAPK axis. Consequently, a cell-free cartilage regeneration strategy using apoVs combined with a decellularized cartilage extracellular matrix (DCM) scaffold effectively promoted the regeneration of osteochondral defects. Overall, new mechanisms of cartilage regeneration by transplanted MSCs were unconcealed in this study. Moreover, we provided a novel experimental basis for cell-free tissue engineering-based cartilage regeneration utilizing apoVs.

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

Incorporation of Magnesium Ions into an Aptamer-Functionalized ECM Bioactive Scaffold for Articular Cartilage Regeneration.

The regeneration and reconstruction of articular cartilage (AC) after a defect are often difficult. The key to the treatment of AC defects lies in regeneration of the defect site and regulation of the inflammatory response. In this investigation, a bioactive multifunctional scaffold was formulated using the aptamer Apt19S as a mediator for mesenchymal stem cell (MSC)-specific recruitment and the enhancement of cellular chondrogenic and inflammatory regulation through the incorporation of Mg2+. Apt19S, which can recruit MSCs in vitro and in vivo, was chemically conjugated to a decellularized cartilage extracellular matrix (ECM)-lysed scaffold. The results from in vitro experiments using the resulting scaffold demonstrated that the inclusion of Mg2+ could stimulate not only the chondrogenic differentiation of synovial MSCs but also the increased polarization of macrophages toward the M2 phenotype. Additionally, Mg2+ inhibited NLRP3 inflammasome activation, thereby decreasing chondrocyte pyroptosis. Subsequently, Mg2+ was incorporated into the bioactive multifunctional scaffold, and the resulting scaffold promoted cartilage regeneration in vivo. In conclusion, this study confirms that the combination of Mg2+ and aptamer-functionalized ECM scaffolds is a promising strategy for AC regeneration based on in situ tissue engineering and early inflammatory regulation.

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure.

Finding an ideal scaffold is always an important issue in the field of cartilage tissue engineering. Both decellularized extracellular matrix and silk fibroin have been used as natural biomaterials for tissue regeneration. In this study, a secondary crosslinking method of γ irradiation and ethanol induction was used to prepare decellularized cartilage extracellular matrix and silk fibroin (dECM-SF) hydrogels with biological activity. Furthermore, the dECM-SF hydrogels were cast in custom-designed molds to produce a three-dimensional multi-channeled structure to improve internal connectivity. The adipose-derived stromal cells (ADSC) were seeded on the scaffolds, cultured in vitro for 2 weeks, and implanted in vivo for another 4 and 12 weeks. The double crosslinked dECM-SF hydrogels exhibited an excellent pore structure after lyophilization. The multi-channeled hydrogel scaffold presents higher water absorption ability, surface wettability, and no cytotoxicity. The addition of dECM and a channeled structure could promote chondrogenic differentiation of ADSC and engineered cartilage formation, confirmed by H&E, safranin O staining, type II collagen immunostaining, and qPCR assay. In conclusion, the hydrogel scaffold fabricated by the secondary crosslinking method has good plasticity and can be used as a scaffold for cartilage tissue engineering. The multi-channeled dECM-SF hydrogel scaffolds possess a chondrogenic induction activity that promotes engineered cartilage regeneration of ADSC in vivo.

Application of tissue-derived bioink for articular cartilage lesion repair

Articular cartilage (AC) lesions are common but remain a great challenge for researchers and clinicians mainly due to their poor self-healing capability. Decellularized cartilage extracellular matrix (dCECM) derived from natural cartilage, and the incorporated growth factors have been used for potential chondrocyte induction and cartilage tissue regeneration. Moreover, infrapatellar fat pad adipose-derived stem cells (IPFP-ADSCs) have been proven to be an optimal source of seeded cells for AC lesion restoration. Therefore, the therapeutic role of dCECM-incorporated bioink with IPFP-ADSCs loaded in 3D (three-dimensional) scaffolds remain to be explored using an osteochondral defect model. In this work, we successfully extracted IPFP-ADSCs and isolated dCECMs, which were then mixed in a temperature-responsive hydrogel to form a 3D bioink. The 3D bioprinting scaffolds printed with dCECM successfully facilitated chondrogenic differentiation-related growth factor expression in IPFP-ADSCs. Most importantly, implantation of the 3D dCECMs with an IPFP-ADSC-loaded scaffold was capable of providing a favorable cell matrix for stem cell proliferation and promoted chondrogenic differentiation, thereby greatly enhancing successful cartilage repair in rabbit cartilage defects.

Microenvironmentally optimized 3D-printed TGFβ-functionalized scaffolds facilitate endogenous cartilage regeneration in sheep

Clinically, microfracture is the most commonly applied surgical technique for cartilage defects. However, an increasing number of studies have shown that the clinical improvement remains questionable, and the reason remains unclear. Notably, recent discoveries revealed that signals from regenerated niches play a critical role in determining mesenchymal stem cell fate specification and differentiation. We speculate that a microenvironmentally optimized scaffold that directs mesenchymal stem cell fate will be a good therapeutic strategy for cartilage repair. Therefore, we first explored the deficiency of microfractures in cartilage repair. The microfracture not only induced inflammatory cell aggregation in blood clots but also consisted of loose granulation tissue with increased levels of proteins related to fibrogenesis. We then fabricated a functional cartilage scaffold using two strong bioactive cues, transforming growth factor-β3 and decellularized cartilage extracellular matrix, to modulate the cell fate of mesenchymal stem cells. Additionally, poly(ε-caprolactone) was also coprinted with extracellular matrix-based bioinks to provide early mechanical support. The in vitro studies showed that microenvironmentally optimized scaffolds exert powerful effects on modulating the mesenchymal stem cell fate, such as promoting cell migration, proliferation and chondrogenesis. Importantly, this strategy achieved superior regeneration in sheep via scaffolds with biomechanics (restored well-organized collagen orientation) and antiapoptotic properties (cell death-related genes were also downregulated). In summary, this study provides evidence that microenvironmentally optimized scaffolds improve cartilage regeneration in situ by regulating the microenvironment and support further translation in human cartilage repair. Statement of significanceAlthough microfracture (MF)-based treatment for chondral defects has been commonly used, critical gaps exist in understanding the biochemistry of MF-induced repaired tissue. More importantly, the clinically unsatisfactory effects of MF treatment have prompted researchers to focus on tissue engineering scaffolds that may have sufficient therapeutic efficacy. In this manuscript, a 3D printing ink containing cartilage tissue-specific extracellular matrix (ECM), methacrylate gelatin (GelMA), and transforming growth factor-β3 (TGF-β3)-embedded polylactic-coglycolic acid (PLGA) microspheres was coprinted with poly(ε-caprolactone) (PCL) to fabricate tissue engineering scaffolds for chondral defect repair. The sustained release of TGF-β3 from scaffolds successfully directed endogenous stem/progenitor cell migration and differentiation. This microenvironmentally optimized scaffold produced improved tissue repair outcomes in the sheep animal model, explicitly guiding more organized neotissue formation and therefore recapitulating the anisotropic structure of native articular cartilage. We hypothesized that the cell-free scaffolds might improve the clinical applicability and become a new therapeutic option for chondral defect repair.

Articular cartilage reconstruction with TGF-β1-simulating self-assembling peptide hydrogel-based composite scaffold

Transforming growth factor-β (TGF-β) is an important inducing factor for the differentiation of mesenchymal stem cells and the secretion of collagen II, but the inaccessibility and instability limit its application in clinical practice. In this study, the TGF-β1-simulating peptide LIANAK (CM) was connected with the self-assembling peptide Ac-(RADA)4-CONH2 (RAD) to obtain the functionalized self-assembling peptide Ac-(RADA)4-GG-LIANAK-CONH2 (RAD-CM). The results indicated that the CM-functionalized RAD hydrogel contributed to the enhanced expressions of chondrogenic genes and extracellular matrix deposition. The self-assembling peptides were then combined with decellularized cartilage extracellular matrix (DCM) to construct a composite scaffold for articular cartilage repair. The CM-functionalized composite scaffold RAD/RAD-CM/DCM (R/C/D) exhibited good bioactivity and structural stability and exhibited satisfactory performance in promoting neocartilage restoration and the reconstruction of the osteochondral unit. This study provides a promising strategy for in situ cartilage regeneration via the stable presentation of TGF-β1-simulating peptide. Statement of significanceDeficiency of effective chondrogenic inducers (especially, the TGF-β family) significantly limits the self-regeneration of cartilage in osteochondral defect cases. Oligopeptide LIANAK, named CM, could simulate TGF-β1’s bioactivity with particular structure, but traditional chemical crosslinking to polymer scaffolds resulted in risks of safety and complication, which is unfavorable for clinical applications. Here, self-assembling peptide RAD was used to load CM, to obtain a TGF-β1 mimetic peptide hydrogel. Depending on the homology (amino acids) of RAD and CM, the synthesis of the whole peptide only needs simply extended sequences of CM following that of RAD by automated solid-phase peptide synthesis. The modified peptide effectively demonstrated osteochondrogenic bioactivity, ensured the convenience, safety, and mass production, which displayed great potential in tissue engineering research and translational medicine.

Mg-BGNs/DCECM Composite Scaffold for Cartilage Regeneration: A Preliminary In Vitro Study.

Cartilage lesions can lead to progressive cartilage degeneration; moreover, they involve the subchondral bone, resulting in osteoarthritis (OA) onset and progression. Bioactive glasses, with the dual function of supporting both bone and cartilage regeneration, have become a promising biomaterial for cartilage/bone engineering applications. This is especially true for those containing therapeutic ions, which act as ion delivery systems and may further promote cartilage repair. In this study, we successfully fabricated Mg-containing bioactive glass nanospheres (Mg-BGNs) and constructed three different scaffolds, DCECM, Mg-BGNs-1/DCECM (1% Mg-BGNs), and Mg-BGNs-2/DCECM (10% Mg-BGNs) scaffold, by incorporating Mg-BGNs into decellularized cartilage extracellular matrix (DCECM). All three scaffolds showed favorable microarchitectural and ion controlled-release properties within the ideal range of pore size for tissue engineering applications. Furthermore, all scaffolds showed excellent biocompatibility and no signs of toxicity. Most importantly, the addition of Mg-BGNs to the DCECM scaffolds significantly promoted cell proliferation and enhanced chondrogenic differentiation induction of mesenchymal stem cells (MSCs) in pellet culture in a dose-dependent manner. Collectively, the multifunctional Mg-BGNs/DCECM composite scaffold not only demonstrated biocompatibility but also a significant chondrogenic response. Our study suggests that the Mg-BGNs/DCECM composite scaffold would be a promising tissue engineering tool for osteochondral lesions, with the ability to simultaneously stimulate articular cartilage and subchondral bone regeneration.

Ex Vivo and In Vivo Properties of an Injectable Hydrogel Derived From Acellular Ear Cartilage Extracellular Matrix.

Extracellular matrix (ECM) hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the adequate bioactivity of native matrix. In this study, we developed decellularized cartilage ECM (dcECM) hydrogels from porcine ears innovatively via the main method of enzymatic digestion and verified good biocompatible properties of dcECM hydrogels to deliver chondrocytes and form subcutaneous cartilage in vivo. The scanning electron microscopy and turbidimetric gelation kinetics were used to characterize the material properties and gelation kinetics of the dcECM hydrogels. Then we evaluated the biocompatibility of hydrogels via the culture of chondrocytes in vitro. To further explore the dcECM hydrogels in vivo, grafts made from the mixture of dcECM hydrogels and chondrocytes were injected subcutaneously in nude mice for the gross and histological analysis. The structural and gelation kinetics of the dcECM hydrogels altered according to the variation in the ECM concentrations. The 10 mg/ml dcECM hydrogels could support the adhesion and proliferation of chondrocytes in vitro. In vivo, at 4 weeks after transplantation, cartilage-like tissues were detected in all groups with positive staining of toluidine blue, Safranin O, and collagen II, indicating the good gelation of dcECM hydrogels. While with the increasing concentration, the tissue engineering cartilages formed by 10 mg/ml dcECM hydrogel grafts were superior in weights, volumes, collagen, and glycosaminoglycan (GAG) content compared to the dcECM hydrogels of 1 mg/ml and 5 mg/ml. At 8 weeks after grafting, dcECM hydrogel grafts at 10 mg/ml showed very similar qualities to the control, collagen I grafts. After 12 weeks of in vivo culture, the histological analysis indicated that 10 mg/ml dcECM hydrogel grafts were similar to the normal cartilage from pig ears, which was the source tissue. In conclusion, dcECM hydrogel showed the promising potential as a tissue engineering biomaterial to improve the regeneration and heal injuries of ear cartilage.

DCECM/DBM-Radmscs Composite Scaffold Acts as a Biosynthetic Keratoprosthesis Skirt with High Biocompatability for Corneal Transplantation

An ideal keratoprosthesis scaffold should have good mechanical property, high biocompatibility, suitable corrosion and wear resistance. Due to limitation of materials, the difference of tenacity between the keratoprosthesis and the cornea often causes the exposure of keratoprosthesis materials, which results in serious complications. The cartilage has the similar biological characteristics to the cornea, no blood vessels and has certain flexibility. Although using the auricular cartilage or rib cartilage to reinforce the keratoprosthesis has been proven greatly successful, it is not possible to be individually designed and idealized. It is urgent to design a biosynthetic material for cartilage replacement to deal with predicament. We prepared and combined the decellularized cartilage extracellular matrix (DCECM) and the decellularized bone matrix (DBM), added the rabbit adipose-derived mesenchymal stem cells (rADMSCs) to construct a biosynthetic keratoprosthesis skirt. We asssessed the mechanical property, cell binding efficiency, safety and cytotoxicity, biocompatability and chondrogenic or osteogenic capacity of the DCECM/DBM-rADMSCs composite scaffold. Tests revealed the DCECM/DBM scaffold has proper elasticity, tenacity, and, is safe to cells. It’s observed that the DCECM/DBM-rADMSCs composite scaffold integrated into the recipient cornea without inflammation and rejection, while transplanted in the recipient cornea for 12 months. In particular, chondrogenesis and osteogenesis are found inside and even around the scaffold. Hence, the DCECM/DBM-rADMSCs composite scaffold coule be a potential biosynthetic skirt for keratoprosthesis or a fixed supportive scaffold to reinforce the keratoprosthesis in the treatment of end-stage corneal blindness.

3D-Bioprinted Difunctional Scaffold for In Situ Cartilage Regeneration Based on Aptamer-Directed Cell Recruitment and Growth Factor-Enhanced Cell Chondrogenesis.

Articular cartilage (AC) lesions are fairly common but remain an obstacle for clinicians and researchers due to their poor self-healing capacity. Recently, a promising therapy based on the recruitment of autologous mesenchymal stem cells (MSCs) has been developed for the regeneration of full-thickness cartilage defects in the knee joint. In this study, a 3D-bioprinted difunctional scaffold was developed based on aptamer HM69-mediated MSC-specific recruitment and growth factor-enhanced cell chondrogenesis. The aptamer, which can specifically recognize and recruit MSCs, was first chemically conjugated to the decellularized cartilage extracellular matrix and then mixed with gelatin methacrylate to form a photocrosslinkable bioink ready for 3D bioprinting. Together with the growth factor that promoted cell chondrogenic differentiation, the biodegradable polymer poly(ε-caprolactone) was further chosen to impart mechanical strength to the 3D bioprinted constructs. The difunctional scaffold specifically recruited MSCs, provided a favorable microenvironment for cell adhesion and proliferation, promoted chondrogenesis, and thus greatly improved cartilage repair in rabbit full-thickness defects. In conclusion, this study demonstrated that 3D bioprinting of difunctional scaffolds could be a promising strategy for in situ AC regeneration based on aptamer-directed cell recruitment and growth-factor-enhanced cell chondrogenesis.

Decellularized Cartilage Extracellular Matrix Incorporated Silk Fibroin Hybrid Scaffolds for Endochondral Ossification Mediated Bone Regeneration

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC’s) via the endochondral ossification (ECO) route. hBMSC’s were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG’s were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC’s under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. μCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC’s expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC’S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.

Cell-free decellularized cartilage extracellular matrix scaffolds combined with interleukin 4 promote osteochondral repair through immunomodulatory macrophages: In vitro and in vivo preclinical study

Cartilage regeneration is a complex physiological process. Synovial macrophages play a critical immunomodulatory role in the acute inflammatory response surrounding joint injury. Due to the contrasting differences and heterogeneity of macrophage, the phenotype of macrophages are the key determinants of the healing response after cartilage injury. Biomaterials derived from extracellular matrix have been used for the repair and reconstruction of a variety of tissues by modulating the host macrophage response. However, the immunomodulatory effect of decellularized cartilage extracellular matrix (ECM) on macrophages has not been elucidated. It is necessary to clarify the immunomodulatory properties of decellularized cartilage matrix (DCM) to guide the design of cartilage regeneration materials. Here, we prepared porcine articular cartilage derived DCM and determined the response of mouse bone marrow-derived macrophages (BMDMs) to the pepsin-solubilized DCM (PDCM) in vitro. Macrophages activated by the PDCM could promote bone marrow-derived mesenchymal stem cells (BMSCs) invasion, migration, proliferation, and chondrogenic differentiation. Then, we verified that early optimization of the immunomodulatory effects of the cell-free DCM scaffold using IL-4 in vivo could achieve good cartilage regeneration in a rat knee osteochondral defect model. Therefore, this decellularized cartilage ECM scaffold combined with accurate and active immunomodulatory strategies provides a new approach for the development of cartilage regeneration materials. Statement of significanceThis work reports a decellularized cartilage extracellular matrix (DCM) scaffold combined with an accurate and active immunomodulatory strategy to improve cartilage regeneration. Our findings demonstrated that the pepsin-solubilized DCM (PDCM) activated bone marrow-derived macrophages to polarize to a constructive macrophage phenotype. These polarized macrophages promoted bone marrow-derived mesenchymal stem cell invasion, migration, proliferation, and chondrogenic differentiation. DCM scaffolds combined with early-stage intra-articular injection of IL-4 created a wound-healing microenvironment and improved cartilage regeneration in a rat knee osteochondral defect model.

Hierarchical macro-microporous WPU-ECM scaffolds combined with Microfracture Promote in Situ Articular Cartilage Regeneration in Rabbits

Tissue engineering provides a promising avenue for treating cartilage defects. However, great challenges remain in the development of structurally and functionally optimized scaffolds for cartilage repair and regeneration. In this study, decellularized cartilage extracellular matrix (ECM) and waterborne polyurethane (WPU) were employed to construct WPU and WPU-ECM scaffolds by water-based 3D printing using low-temperature deposition manufacturing (LDM) system, which combines rapid deposition manufacturing with phase separation techniques. The scaffolds successfully achieved hierarchical macro‐microporous structures. After adding ECM, WPU scaffolds were markedly optimized in terms of porosity, hydrophilia and bioactive components. Moreover, the optimized WPU-ECM scaffolds were found to be more suitable for cell distribution, adhesion, and proliferation than the WPU scaffolds. Most importantly, the WPU-ECM scaffold could facilitate the production of glycosaminoglycan (GAG) and collagen and the upregulation of cartilage-specific genes. These results indicated that the WPU-ECM scaffold with hierarchical macro‐microporous structures could recreate a favorable microenvironment for cell adhesion, proliferation, differentiation, and ECM production. In vivo studies further revealed that the hierarchical macro‐microporous WPU-ECM scaffold combined with the microfracture procedure successfully regenerated hyaline cartilage in a rabbit model. Six months after implantation, the repaired cartilage showed a similar histological structure and mechanical performance to that of normal cartilage. In conclusion, the hierarchical macro‐microporous WPU-ECM scaffold may be a promising candidate for cartilage tissue engineering applications in the future.

3D-Printed Extracellular Matrix/Polyethylene Glycol Diacrylate Hydrogel Incorporating the Anti-inflammatory Phytomolecule Honokiol for Regeneration of Osteochondral Defects

Background: Osteoarthritis is the leading cause of disability worldwide; cartilage degeneration and defects are the central features. Significant progress in tissue engineering holds promise to regenerate damaged cartilage tissue. However, a formidable challenge is to develop a 3-dimensional (3D) tissue construct that can regulate local immune environment to facilitate the intrinsic osteochondral regeneration. Purpose: To evaluate efficacy of a 3D-printed decellularized cartilage extracellular matrix (ECM) and polyethylene glycol diacrylate (PEGDA) integrated novel scaffold (PEGDA/ECM) together with the natural compound honokiol (Hon) for regenerating osteochondral defect. Study Design: Controlled laboratory study. Methods: We used a stereolithography-based 3D printer for PEGDA/ECM bioprinting. A total of 36 Sprague-Dawley rats with cylindrical osteochondral defect in the trochlear groove of the femur were randomly assigned into 3 different treatments: no scaffold implantation (Defect group), 3D printed PEGDA/ECM scaffold alone (PEGDA/ECM group), or Hon suspended in a 3D-printed PEGDA/ECM scaffold (PEGDA/ECM/Hon group). 12 rats that underwent only medial parapatellar incision surgery were used as normal controls. The femur specimens were postoperatively harvested at 4 and 8 weeks for gross, micro-CT, and histological evaluations. The efficacy of PEGDA/ECM/Hon scaffold on the release of proinflammatory cytokines from the macrophages stimulated by lipopolysaccharide (LPS) was evaluated in-vitro. Results: In vitro results determined that PEGDA/ECM/Hon scaffold could suppress the release of proinflammatory cytokines from macrophages that were stimulated by LPS. Macroscopic images showed that the PEGDA/ECM/Hon group had significantly higher ICRS scoring than that of defect and PEGDA/ECM groups. Micro-CT evaluation demonstrated that much more bony tissue was formed in the defect sites implanted with the PEGDA/ECM scaffold or PEGDA/ECM/Hon scaffold compared with the untreated defects. Histological analysis showed that the PEGDA/ECM/Hon group had a significant enhancement in osteochondral regeneration at 4 and 8 weeks after surgery in comparison with the ECM/PEGDA or defect group. Conclusion: This study demonstrated that 3D printing of PEGDA/ECM hydrogel incorporating the anti-inflammatory phytomolecule honokiol could provide a promising scaffold for osteochondral defect repair.

Three-Dimensional Porous Scaffolds with Biomimetic Microarchitecture and Bioactivity for Cartilage Tissue Engineering.

Ideal tissue-engineering cartilage scaffolds should possess the same nanofibrous structure as the microstructure of native cartilage as well as the same biological function provided by the microenvironment for neocartilage regeneration. In the present study, three-dimensional composite biomimetic scaffolds with different concentration ratios of electrospun gelatin-polycaprolactone (gelatin-PCL) nanofibers and decellularized cartilage extracellular matrix (DCECM) were fabricated. The nanofibers with the biomimetic microarchitecture of native cartilage served as a skeleton with excellent mechanical properties, and the DCECM served as a biological functionalization platform for the induction of cell response and the promotion of cartilage regeneration. Experimental results showed that the composite nanofiber/DCECM (NF/DCECM) scaffolds had stronger mechanical properties and structural stability in wet state compared with those of DCECM scaffolds. In vitro experiments demonstrated that all scaffolds had good biocompatibility, but the chondrocyte proliferation rate of the composite NF/DCECM scaffolds was higher than that of the NF scaffolds. In vitro and in vivo cartilage regeneration results indicated that the DCECM component of the composite scaffolds facilitated early maturation of the cartilage lacuna and the secretion of collagen and glycosaminoglycan. The macroscopic and histological results at 12 weeks postsurgery exhibited that the composite NF/DCECM scaffolds yielded better cartilage repair outcomes than those of the nontreated group and NF scaffolds group. Overall, the present study demonstrated that the structurally and functionally biomimetic NF/DCECM scaffold is a promising tissue engineering scaffold for cartilage regeneration and cartilage defect repair.

Fluorescent Nanodiamonds Enable Long-Term Detection of Human Adipose-Derived Stem/Stromal Cells in an In Vivo Chondrogenesis Model Using Decellularized Extracellular Matrices and Fibrin Glue Polymer.

Clinically available materials, including allogeneic irradiated costal cartilage and fibrin glue polymer, were used as scaffolds for in vivo chondrogenic differentiation of human adipose-derived stem/stromal cells (hASCs) in the attempt to develop a more efficient treatment over current methods. Current studies include the use of growth-factor stimulation, tissue engineering, and biocompatible materials; however, most methods involve complicated processes and pose clinical limitations. In this report, the xenografts in the experimental group composed of a diced decellularized cartilage extracellular matrix (ECM), hASCs, and fibrin glue polymer were implanted into the subcutaneous layer of nude mice, and the results were compared with two groups of controls; one control group received implantation of decellularized cartilage ECM and fibrin glue polymer, and the other control group received implantation of hASCs mixed with fibrin glue polymer. To evaluate whether hASCs had in vivo chondrogenesis in the xenografts, hASCs were labeled with fluorescent nanodiamonds (FNDs), a biocompatible and photostable nanomaterial, to allow for long-term detection and histological analysis. Increased cellularity, glycosaminoglycan, and collagen deposition were found by the histological examination in the experimental group compared with control groups. With the background-free detection technique and time-gated fluorescence imaging, the numbers and locations of the FND-labeled hASCs could be detected by confocal microscopy. The chondrocyte-specific markers, such as aggrecan and type II collagen, were colocalized with cells containing signals of FNDs which indicated in vivo chondrogenesis of hASCs. Taken together, functional in vivo chondrogenesis of the hASCs could be achieved by clinically available decellularized cartilage ECM and fibrin glue polymer in the nude mice model without in vitro chondrogenic induction. The fluorescent signals of FNDs in hASCs can be detected in histological analysis, such as hematoxylin and eosin staining (H&E staining) without the interference of the autofluorescence. Our study may warrant future clinical applications of the combination of decellular cartilage ECM, fibrin glue polymer, and hASCs for cartilage repair.

Silk fibroin/cartilage extracellular matrix scaffolds with sequential delivery of TGF-β3 for chondrogenic differentiation of adipose-derived stem cells.

A 3-D scaffold that simulates the microenvironment in vivo for regenerating cartilage is ideal. In this study, we combined silk fibroin and decellularized cartilage extracellular matrix by temperature gradient-guided thermal-induced phase separation to produce composite scaffolds (S/D). Resulting scaffolds had remarkable mechanical properties and biomimeticstructure, for a suitable substrate for attachment and proliferation of adipose-derived stem cells (ADSCs). Moreover, transforming growth factor β3 (TGF-β3) loaded on scaffolds showed a controlled release profile and enhanced the chondrogenic differentiation of ADSCs during the 28-day culture. The S/D scaffold itself can provide a sustained release system without the introduction of other controlled release media, which has potential for commercial and clinical applications. The results of toluidine blue, Safranin O, and immunohistochemical staining and analysis of collagen II expression showed maintenance of a chondrogenic phenotype in all scaffolds after 28-day culture. The most obvious phenomenon was with the addition of TGF-β3. S/D composite scaffolds with sequential delivery of TGF-β3 may mimic the regenerative microenvironment to enhance the chondrogenic differentiation of ADSCs in vitro.

Ultrastructure of bioscaffolds derived from bovine articular cartilage as an experimental model to support blastema cells

Decellularized cartilage extracellular matrix (ECM)-derived bioscaffolds have been designed as they provide a unique opportunity to carry out research on the mechanisms of chondrogenesis and its regulation. These scaffolds are used as a model in order to emulate different aspects relating to the formation, degeneration, and regeneration of the cartilage. This project has studied the interaction between the decellularized bovine articular cartilage and the blastema cells derived from the punched rabbit’s ear. Articular cartilage was dissected in fragments with 10 mm length and 2 mm thickness. Then, they were chemically decellularized with 2% sodium dodecyl sulfate (SDS) for 2 h. The tissue rings derived from the rabbit’s ear were assembled with the decellularized scaffolds and they were placed in a culture media for 30 days. These samples were fixed, sectioned, and microscopically studied. Micrographs of SEM electron microscopy and DAPI staining confirmed the decellularization of the scaffolds. FTIR analysis confirmed the preservation of ECM components including collagen II and proteoglycans. The optical microscopy observations confirmed migration, adherence, and penetration of the blastema cells into the scaffolds. The electron microscopy studies demonstrated that the blastema cells beside the scaffolds have triggered mutual interactions and merely progressed toward chondrogenic differentiation. The main objective is to identify and comprehend the interaction between cartilage matrix and blastema cells. The proposed model is an ideal model for fundamental studies in cartilage tissue engineering. The principles of tissue engineering must be taken in to account while studying such interactions.