[Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology].

Objective: To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods: The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results: The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion: The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.

Meniscal repair with additive manufacture of bioresorbable polymer: From physicochemical characterization to implantation of 3D printed poly (L-co-D, L lactide-co-trimethylene carbonate) with autologous stem cells in rabbits.

Three-dimensional (3D) structures are actually the state-of-the-art technique to create porous scaffolds for tissue engineering. Since regeneration in cartilage tissue is limited due to intrinsic cellular properties this study aims to develop and characterize three-dimensional porous scaffolds of poly (L-co-D, L lactide-co-trimethylene carbonate), PLDLA-TMC, obtained by 3D fiber deposition technique. The PLDLA-TMC terpolymer scaffolds (70:30), were obtained and characterized by scanning electron microscopy, gel permeation chromatography, differential scanning calorimetry, thermal gravimetric analysis, compression mechanical testing and study on in vitro degradation, which showed its amorphous characteristics, cylindrical geometry, and interconnected pores. The in vitro degradation study showed significant loss of mechanical properties compatible with a decrease in molar mass, accompanied by changes in morphology. The histocompatibility association of mesenchymal stem cells from rabbit's bone marrow, and PLDLA-TMC scaffolds, were evaluated in the meniscus regeneration, proving the potential of cell culture at in vivo tissue regeneration. Nine New Zealand rabbits underwent total medial meniscectomy, yielding three treatments: implantation of the seeded PLDLA-TMC scaffold, implantation of the unseeded PLDLA-TMC and negative control (defect without any implant). After 24 weeks, the results revealed the presence of fibrocartilage in the animals treated with polymer. However, the regeneration obtained with the seeded PLDLA-TMC scaffolds with mesenchymal stem cells had become intimal to mature fibrocartilaginous tissue of normal meniscus both macroscopically and histologically. This study demonstrated the effectiveness of the PLDLA-TMC scaffold in meniscus regeneration and the potential of mesenchymal stem cells in tissue engineering, without the use of growth factors. It is concluded that bioresorbable polymers represent a promising alternative for tissue regeneration.

Tricomposite gelatin-carboxymethylcellulose-alginate bioink for direct and indirect 3D printing of human knee meniscal scaffold.

The development of technologies that could ease the production of customizable patient-specific tissue engineering constructs having required biomechanical properties and restoring function in damaged tissue is the need of the hour. In this study, we report the optimization of composite, bioactive and biocompatible tripolymeric hydrogel bioink, suitable for both direct and indirect printing of customizable scaffolds for cartilage tissue engineering applications. A customized hierarchical meniscal scaffold was designed using solid works software and developed using a negative mould made of polylactic acid (PLA) filament and by a direct 3D printing process. A composite tripolymeric bioink made of gelatin, carboxymethyl cellulose (CMC) and alginate was optimized and characterized for its printability, structural, bio-mechanical and bio-functional properties. The optimized composite hydrogel bioink was extruded into the negative mould with and without live cells, cross-linked and the replica of meniscus structure was retrieved aseptically. The cellular proliferation, apatite formation, and extracellular matrix secretion from negative printed meniscal scaffold were determined using MTT, live/dead and collagen estimation assays. A significant increase in collagen secretion, cellular proliferation and changes in biomechanical properties was observed in the 3D scaffolds with MG63-osteosarcoma cells indicating its suitability for cartilage tissue engineering.

Precision 3D printed meniscus scaffolds to facilitate hMSCs proliferation and chondrogenic differentiation for tissue regeneration.

BACKGROUND: The poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue. RESULTS: Herein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility. CONCLUSIONS: Overall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration.

Potential of Melt Electrowritten Scaffolds Seeded with Meniscus Cells and Mesenchymal Stromal Cells.

Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants® (CMI®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young's modulus when compared to the CMI® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment.

Selection of Highly Proliferative and Multipotent Meniscus Progenitors through Differential Adhesion to Fibronectin: A Novel Approach in Meniscus Tissue Engineering.

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.

Anti-Inflammatory Effects of Conditioned Medium of Periodontal Ligament-Derived Stem Cells on Chondrocytes, Synoviocytes, and Meniscus Cells.

Osteoarthritis (OA) is the most common type of arthritis, afflicting millions of people in the world. Elevation of inflammatory mediators and enzymatic matrix destruction is often associated with OA. Therefore, the objective of this study was to investigate the effects of conditioned medium from periodontal ligament-derived stem cells (PDLSCs) on inflammatory and catabolic gene expressions of chondrocytes, synoviocytes, and meniscus cells under in vitro inflammatory condition. Stem cells were isolated from human periodontal ligaments. Conditioned medium was collected and concentrated 20 × . Chondrocytes, synoviocytes, and meniscus cells were isolated from pig knees and divided into four experimental groups: serum-free media, serum-free media+interleukin-1ß (IL-1ß) (10 ng/mL), conditioned media (CM), and CM+IL-1ß. Protein content and extracellular vesicle (EV) miRNAs of CM were analyzed by liquid chromatography-tandem mass spectrometry and RNA sequencing, respectively. It was found that the IL-1ß treatment upregulated the expression of IL-1ß, tumor necrosis factor-α (TNF-α), MMP-13, and ADAMTS-4 genes in the three cell types, whereas PDLSC-conditioned medium prevented the upregulation of gene expression by IL-1ß in all three cell types. This study also found that there was consistency in anti-inflammatory effects of PDLSC CM across donors and cell subcultures, while PDLSCs released several anti-inflammatory factors and EV miRNAs at high levels. OA has been suggested as an inflammatory disease in which all intrasynovial tissues are involved. PDLSC-conditioned medium is a cocktail of trophic factors and EV miRNAs that could mediate different inflammatory processes in various tissues in the joint. Introducing PDLSC-conditioned medium to osteoarthritic joints could be a potential treatment to prevent OA progression by inhibiting inflammation.

Gene expression profiling of primary fibrochondrocyte cultures in traumatic and degenerative meniscus lesions.

PURPOSE: This study aimed to investigate how fibroblastic and chondrocytic properties of human meniscal fibrochondrocytes are affected in culture conditions according to the type of meniscal pathology and localization, and to provide basic information for tissue-engineering studies. METHODS: Primary fibrochondrocyte cultures were prepared from meniscus samples of patients who had either traumatic tear or degeneration due to osteoarthritis. Cultures were compared in terms of mRNA expression levels of COL1A1, COL2A1, COMP1, HIF1A, HIF2A, and SOX9 and secreted total collagen and sulfated sGAG levels according to the type of meniscal pathology, anatomical localization, and the number of subcultures. RESULTS: mRNA expression levels of COL1A1, COMP1, HIF1A, HIF2A, and SOX9 were found to be increased in subsequent subcultures in all specimens. COL1A1 mRNA expression levels of both lateral and medial menisci of patients with traumatic tear were significantly higher than in patients with degenerative pathology, indicating a more fibroblastic character. P1 subculture of lateral and P3 or further subculture of medial meniscus showed more fibroblastic characteristics in patients with degenerative pathology. Furthermore, in patients with degenerative pathology, the subcultures of the lateral meniscus (especially on the inner part) presented more chondrocytic characteristics than did those of medial meniscus. CONCLUSIONS: The mRNA expression levels of the cultures showed significant differences according to the anatomical localization and pathology of the meniscus, indicating distinct chondrocytic and fibroblastic features. This fundamental knowledge would help researchers to choose more efficient cell sources for cell-seeding of a meniscus scaffold, and to generate a construct resembling the original meniscus tissue.

Degeneration of the articular disc in the human triangular fibrocartilage complex.

INTRODUCTION: Traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent reasons for ulnar wrist pain. The assessment of the extent of articular disc (AD) degeneration is important for the differentiation of acute injuries versus chronic lesions. MATERIALS AND METHODS: The AD of the TFCC of eleven human cadaver wrists was dissected. Degeneration was analyzed according to the grading of Krenn et al. Hematoxylin-eosin was used to determine the tissue morphology. Degeneration was evaluated using the staining intensity of alcian blue, the immunohistochemistry of the proteoglycan versican and the immunoreactivity of NITEGE, an aggrecan fragment. RESULTS: The staining homogeneity of HE decreased with higher degeneration of the AD and basophilic tissue areas were more frequently seen. Two specimens were characterized as degeneration grade 1, five specimens as grade 2, and four specimens as grade 3, respectively. Staining intensity of alcian blue increased with higher degeneration grade of the specimens. Immunoreactivity for NITEGE was detected around tissue fissures and perforations as well as matrix splits. Immunoreactivity for versican was found concentrated in the tissue around matrix fissures and lesions as well as loose connective tissue at the ulnar border of the AD. Specimens with degeneration grade 2 had the strongest immunoreactivity of NITEGE and versican. Cell clusters were observed in specimens with degeneration grade 2 and 3, which were stained by alcian blue and immunoreactive for NITEGE and versican. Increasing age of the cadaver wrists correlated with a higher degree of degeneration (p < 0.0001, r = 0.68). CONCLUSIONS: The fibrocartilage of degenerated ADs contains NITEGE and versican. The amount of the immunoreactivity of these markers allows the differentiation of degenerative changes into three grades. The degeneration of the AD increases with age and emphasizes its important mechanical function.

Development of biological meniscus scaffold: Decellularization method and recellularization with meniscal cell population derived from mesenchymal stem cells.

Tissue engineering approaches which include a combination of cells and scaffold materials provide an alternative treatment for meniscus regeneration. Decellularization and recellularization techniques are potential treatment options for transplantation. Maintenance of the ultrastructure composition of the extracellular matrix and repopulation with cells are important factors in constructing a biological scaffold and eliminating immunological reactions.The aim of the study is to develop a method to obtain biological functional meniscus scaffolds for meniscus regeneration. For this purpose, meniscus tissue was decellularized by our modified method, a combination of physical, chemical, and enzymatic methods and then recellularized with a meniscal cell population composed of fibroblasts, chondrocytes and fibrochondrocytes that obtained from mesenchymal stem cells. Decellularized and recellularized meniscus scaffolds were analysed biochemically, biomechanically and histologically. Our results revealed that cellular components of the meniscus were successfully removed by preserving collagen and GAG structures without any significant loss in biomechanical properties. Recellularization results showed that the meniscal cells were localized in the empty lacuna on the decellularized meniscus, and also well distributed and proliferated consistently during the cell culture period (p < 0.05). Furthermore, a high amount of DNA, collagen, and GAG contents (p < 0.05) were obtained with the meniscal cell population in recellularized meniscus tissue.The study demonstrates that our decellularization and recellularization methods were effective to develop a biological functional meniscus scaffold and can mimic the meniscus tissue with structural and biochemical features. We predict that the obtained biological meniscus scaffolds may provide avoidance of adverse immune reactions and an appropriate microenvironment for allogeneic or xenogeneic recipients in the transplantation process. Therefore, as a promising candidate, the obtained biological meniscus scaffolds might be verified with a transplantation experiment.

Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus.

PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Meniscal tissue engineering via 3D printed PLA monolith with carbohydrate based self-healing interpenetrating network hydrogel.

Failure of bioengineered meniscus implant after transplantation is a major concern owing to mechanical failure, lack of chondrogenic capability and patient specific design. In this article, we have, for the first time, fabricated a 3D printed scaffold with carbohydrate based self-healing interpenetrating network (IPN) hydrogels-based monolith construct for load bearing meniscus tissue. 3D printed PLA scaffold was surface functionalized and embedded with self-healing IPN hydrogel for interfacial bonding further characterized by micro CT. Using collagen (C), alginate (A) and oxidized alginate (ADA), we developed self-healing IPN hydrogels with dual crosslinking (Ca2+ based ionic crosslinking and Schiff base (A-A, A-ADA)) capability and studied their physicochemical properties. Further, we studied human stem cells behaviour and chondrogenic differentiation potential within these IPN hydrogels. In-vivo heterotopic implantation confirmed biocompatibility of the monolith showing the feasibility of using carbohydrate based IPN hydrogel embedded in 3D printed scaffold for meniscal tissue development.

Biomechanically, structurally and functionally meticulously tailored polycaprolactone/silk fibroin scaffold for meniscus regeneration.

Meniscus deficiency, the most common and refractory disease in human knee joints, often progresses to osteoarthritis (OA) due to abnormal biomechanical distribution and articular cartilage abrasion. However, due to its anisotropic spatial architecture, complex biomechanical microenvironment, and limited vascularity, meniscus repair remains a challenge for clinicians and researchers worldwide. In this study, we developed a 3D printing-based biomimetic and composite tissue-engineered meniscus scaffold consisting of polycaprolactone (PCL)/silk fibroin (SF) with extraordinary biomechanical properties and biocompatibility. We hypothesized that the meticulously tailored composite scaffold could enhance meniscus regeneration and cartilage protection. Methods: The physical property of the scaffold was characterized by scanning electron microscopy (SEM) observation, degradation test, frictional force of interface assessment, biomechanical testing, and fourier transform infrared (FTIR) spectroscopy analysis. To verify the biocompatibility of the scaffold, the viability, morphology, proliferation, differentiation, and extracellular matrix (ECM) production of synovium-derived mesenchymal stem cell (SMSC) on the scaffolds were assessed by LIVE/DEAD staining, alamarBlue assay, ELISA analysis, and qRT-PCR. The recruitment ability of SMSC was tested by dual labeling with CD29 and CD90 by confocal microscope at 1 week after implantation. The functionalized hybrid scaffold was then implanted into the meniscus defects on rabbit knee joint for meniscus regeneration, comparing with the Blank group (no scaffold) and PS group. The regenerated meniscus tissue was evaluated by histological and immunohistochemistry staining, and biomechanical test. Macroscopic and histological scoring was performed to assess the outcome of meniscus regeneration and cartilage protection in vivo. Results: The combination of SF and PCL could greatly balance the biomechanical properties and degradation rate to match the native meniscus. SF sponge, characterized by fine elasticity and low interfacial shear force, enhanced energy absorption capacity of the meniscus and improved chondroprotection. The SMSC-specific affinity peptide (LTHPRWP; L7) was conjugated to the scaffold to further increase the recruitment and retention of endogenous SMSCs. This meticulously tailored scaffold displayed superior biomechanics, structure, and function, creating a favorable microenvironment for SMSC proliferation, differentiation, and extracellular matrix (ECM) production. After 24 weeks of implantation, the histological assessment, biochemical contents, and biomechanical properties demonstrated that the polycaprolactone/silk fibroin-L7 (PS-L7) group was close to the native meniscus group, showing significantly better cartilage protection than the PS group. Conclusion: This tissue engineering scaffold could greatly strengthen meniscus regeneration and chondroprotection. Compared with traditional cell-based therapies, the meniscus tissue engineering approach with advantages of one-step operation and reduced cost has a promising potential for future clinical and translational studies.

Synovial mesenchymal stem cells effectively alleviate osteoarthritis through promoting the proliferation and differentiation of meniscus chondrocytes.

OBJECTIVE: To investigate the relationship between the meniscal defect area and OA progression and explore the effect and mechanism of SMSCs cell therapy in knee osteoarthritis (OA) rat model. MATERIALS AND METHODS: For animal experiments, knee osteoarthritis (OA) model was constructed in Sprague Dawley (SD) rats by removing the medial meniscus of the right knee. Synovial mesenchymal stem cells (SMSCs) were engrafted by injecting into the right knee cavity. For in vitro experiments, CCK-8 assay was performed to evaluate the proliferation and differentiation of BMSCs and ATDC5 cells after co-cultured with SMSCs. qRT-PCR analysis was performed to detect the expressions of chondrogenic genes in BMSCs and ATDC5 cells after co-cultured with SMSCs. Western blot analysis was conducted to detect the phosphorylations of c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases (ERK) in MAPK signaling of BMSCs and ATDC5 cells. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum levels of interleukin (IL)-1ß, IL-1ß, IL-6, IL-18 and C-reactive protein (CRP). RESULTS: Results showed that meniscus damaged area is positively correlated to serum inflammatory factor levels. In vitro study showed that the proliferation and differentiation of BMSCs and ATDC5 cells were promoted after co-cultured with SMSCs. By co-culturing with SMSCs, the MAPK signaling pathway was activated and the expression of chondrogenic markers such as aggrecan (acan), SRY-related high mobility group-box gene 9 (sox9) and Type II collagen a1 (col2a1), was up-regulated both in BMSCs and ATDC5 cells. In vivo study showed SMSCs cell therapy significantly decreased serum inflammatory factor levels and protected cartilage by upregulating the expression of chondrogenic genes of meniscus chondrocytes derived from OA rats. CONCLUSIONS: For the first time, we found the positive correlation between meniscal defect area and OA progression and demonstrated the effect and mechanism of SMSCs cell therapy in knee osteoarthritis (OA) treatment.

Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone and porous silk fibroin for meniscus tissue engineering.

The meniscus has critical functions in the knee joint kinematics and homeostasis. Injuries of the meniscus are frequent, and the lack of a functional meniscus between the femur and tibial plateau can cause articular cartilage degeneration leading to osteoarthritis development and progression. Regeneration of meniscus tissue has outstanding challenges to be addressed. In the current study, novel Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone (PCL) and porous silk fibroin were proposed for meniscus tissue engineering. As confirmed by micro-structural analysis the entrapment of silk fibroin was successful, and all scaffolds had excellent interconnectivity (≥99%). The EiC scaffolds had more favorable micro-structure compared with the PCL cage scaffolds by improving the pore size while keeping the interconnectivity almost the same. When compared with the PCL cage, the entrapment of porous silk fibroin into the PCL cage decreased the high compressive modulus in a favorable matter in the wet state thanks to the silk fibroin's high swelling properties. The in vitro studies with human stem cells or meniscocytes seeded constructs, demonstrated that the EiC scaffolds had superior cell adhesion, metabolic activity, and proliferation compared to the PCL cage scaffolds. Upon subcutaneous implantation of scaffolds in nude mice, all groups were free of adverse incidents, and mildly invaded by inflammatory cells with neovascularization, while the EiC scaffolds showed better tissue infiltration. The results of this work indicated that the EiC scaffolds of PCL and silk fibroin are favorable for meniscus tissue engineering, and the findings are encouraging for further studies using a larger animal model.

Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration.

OBJECTIVES: The heterogeneity of meniscus cells and the mechanism of meniscus degeneration is not well understood. Here, single-cell RNA sequencing (scRNA-seq) was used to identify various meniscus cell subsets and investigate the mechanism of meniscus degeneration. METHODS: scRNA-seq was used to identify cell subsets and their gene signatures in healthy human and degenerated meniscus cells to determine their differentiation relationships and characterise the diversity within specific cell types. Colony-forming, multi-differentiation assays and a mice meniscus injury model were used to identify meniscus progenitor cells. We investigated the role of degenerated meniscus progenitor (DegP) cell clusters during meniscus degeneration using computational analysis and experimental verification. RESULTS: We identified seven clusters in healthy human meniscus, including five empirically defined populations and two novel populations. Pseudotime analysis showed endothelial cells and fibrochondrocyte progenitors (FCP) existed at the pseudospace trajectory start. Melanoma cell adhesion molecule ((MCAM)/CD146) was highly expressed in two clusters. CD146+ meniscus cells differentiated into osteoblasts and adipocytes and formed colonies. We identified changes in the proportions of degenerated meniscus cell clusters and found a cluster specific to degenerative meniscus with progenitor cell characteristics. The reconstruction of four progenitor cell clusters indicated that FCP differentiation into DegP was an aberrant process. Interleukin 1ß stimulation in healthy human meniscus cells increased CD318+ cells, while TGFß1 attenuated the increase in CD318+ cells in degenerated meniscus cells. CONCLUSIONS: The identification of meniscus progenitor cells provided new insights into cell-based meniscus tissue engineering, demonstrating a novel mechanism of meniscus degeneration, which contributes to the development of a novel therapeutic strategy.

Characterization and Comparison of Postnatal Rat Meniscus Stem Cells at Different Developmental Stages.

Meniscus-derived stem cells (MeSCs) are a potential cell source for meniscus tissue engineering. The stark morphological and structural changes of meniscus tissue during development indicate the complexity of MeSCs at different tissue regions and stages of development. In this study, we characterized and compared postnatal rat meniscus tissue and MeSCs at different tissue regions and stages of development. We observed that the rat meniscus tissue exhibited marked changes in tissue morphology during development, with day 7 being the most representative time point of different developmental stages. All rat MeSCs displayed typical stem cell characteristics. Rat MeSCs derived from day 7 inner meniscus tissue exhibited the highest self-renewal capacity, cell proliferation, differentiation potential toward various mesenchymal lineage and the highest expression levels of chondrogenic genes and proteins. Transplantation of rat MeSCs derived from day 7 inner meniscus tissue promoted neo-tissue formation and effectively protected joint surface cartilage in vivo. Our results demonstrated for the first time that rat MeSCs are not necessarily better at earlier developmental stages, and that rat MeSCs derived from day 7 inner meniscus tissue may be a superior cell source for effective meniscus regeneration and articular cartilage protection. This information could make a significant contribution to human meniscus tissue engineering in the future. Stem Cells Translational Medicine 2019;8:1318&1329.

Meniscus regeneration combining meniscus and mesenchymal stromal cells in a degradable meniscus implant: an in vitro study.

Meniscus regeneration is an unmet clinical need as damage to the meniscus is common and causes early osteoarthritis. The aim of the present study was to investigate the feasibility of a one-stage cell-based treatment for meniscus regeneration by augmenting a resorbable collagen-based implant with a combination of recycled meniscus cells and mesenchymal stromal cells (MSCs). Cell communication and fate of the different cell types over time in co-culture were evaluated by connexin 43 staining for gap junctions and polymerase chain reaction (PCR) to discriminate between meniscus cells and MSCs, based on a Y-chromosome gene. To define optimal ratios, human meniscus cells and bone-marrow-derived MSCs were cultured in different ratios in cell pellets and type I collagen hydrogels. In addition, cells were seeded on the implant in fibrin glue by static seeding or injection. Cellular communication by gap junctions was shown in co-culture and a decrease in the amount of MSCs over time was demonstrated by PCR. 20 : 80 and 10 : 90 ratios showed significantly highest glycosaminoglycan and collagen content in collagen hydrogels. The same statistical trend was found in pellet cultures. Significantly more cells were present in the injected implant and cell distribution was more homogenous as compared to the statically seeded implant. The study demonstrated the feasibility of a new one-stage cell-based procedure for meniscus regeneration, using 20 % meniscus cells and 80 % MSCs seeded statically on the implant. In addition, the stimulatory effect of MSCs towards meniscus cells was demonstrated by communication through gap junctions.

Meniscus-Derived Matrix Scaffolds Promote the Integrative Repair of Meniscal Defects.

Meniscal tears have a poor healing capacity, and damage to the meniscus is associated with significant pain, disability, and progressive degenerative changes in the knee joint that lead to osteoarthritis. Therefore, strategies to promote meniscus repair and improve meniscus function are needed. The objective of this study was to generate porcine meniscus-derived matrix (MDM) scaffolds and test their effectiveness in promoting meniscus repair via migration of endogenous meniscus cells from the surrounding meniscus or exogenously seeded human bone marrow-derived mesenchymal stem cells (MSCs). Both endogenous meniscal cells and MSCs infiltrated the MDM scaffolds. In the absence of exogenous cells, the 8% MDM scaffolds promoted the integrative repair of an in vitro meniscal defect. Dehydrothermal crosslinking and concentration of the MDM influenced the biochemical content and shear strength of repair, demonstrating that the MDM can be tailored to promote tissue repair. These findings indicate that native meniscus cells can enhance meniscus healing if a scaffold is provided that promotes cellular infiltration and tissue growth. The high affinity of cells for the MDM and the ability to remodel the scaffold reveals the potential of MDM to integrate with native meniscal tissue to promote long-term repair without necessarily requiring exogenous cells.

Development of decellularized meniscus extracellular matrix and gelatin/chitosan scaffolds for meniscus tissue engineering.

BACKGROUND: Meniscus tissue engineering has provided a great potential treatment for meniscal injuries. However, few scaffolds in meniscus tissue engineering have matched the mechanical properties of native meniscus. OBJECTIVE: In this study, we developed a composite scaffold using decellularized meniscus extracellular matrix (DMECM) and gelatin/chitosan (G/C) to explore a preferable ratio to enhance the elastic modulus and cytotoxicity properties of scaffolds. METHODS: The microstructure, porosity, cytotoxicity, and strength of the composite scaffolds were evaluated. The micro-architectures of the samples were evaluated using scanning electron microscope (SEM). Fourier Transform Infrared analysis (FTIR) was used to confirm the chemical structure with different type composite scaffolds. The compressive elastic modulus of all the scaffolds were measured by the universal tensile testing machine DNS300. Calcein-AM (fluorescent green) and propidium iodide (fluorescent red) were used to stain live cells and dead cells. Morphology and spatial distribution of cells within scaffolds were observed by confocal laser scanning microscopy FV 1000. RESULTS: SEM showed that the composite scaffolds had suitable porous structure. CCK-8 and live/dead staining demonstrated that the composite scaffolds had no cytotoxicity and could promote bone marrow mesenchymal stem cells (BMSCs) proliferation. The FTIR results demonstrated the successful mixing of these two elements, and the addition of DMECM improved the elastic modulus and cytotoxicity of G/C composite scaffolds. CONCLUSIONS: This study developed a composite scaffold using DMECM and G/C, and demonstrated that it might be suitable for meniscal tissue engineering application.