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.

Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6-CXCR2 pathway.

OBJECTIVE: To investigate the efficacy of basic fibroblast growth factor (bFGF) in promoting meniscus regeneration by cultivating synovial mesenchymal stem cells (SMSCs) and to validate the underlying mechanisms. METHODS: Human SMSCs were collected from patients with osteoarthritis. Eight-week-old nude rats underwent hemi-meniscectomy, and SMSCs in pellet form, either with or without bFGF (1.0 × 106 cells per pellet), were implanted at the site of meniscus defects. Rats were divided into the control (no transplantation), FGF (-) (pellet without bFGF), and FGF (+) (pellet with bFGF) groups. Different examinations, including assessment of the regenerated meniscus area, histological scoring of the regenerated meniscus and cartilage, meniscus indentation test, and immunohistochemistry analysis, were performed at 4 and 8 weeks after surgery. RESULTS: Transplanted SMSCs adhered to the regenerative meniscus. Compared with the control group, the FGF (+) group had larger regenerated meniscus areas, superior histological scores of the meniscus and cartilage, and better meniscus mechanical properties. RNA sequencing of SMSCs revealed that the gene expression of chemokines that bind to CXCR2 was upregulated by bFGF. Furthermore, conditioned medium derived from SMSCs cultivated with bFGF exhibited enhanced cell migration, proliferation, and chondrogenic differentiation, which were specifically inhibited by CXCR2 or CXCL6 inhibitors. CONCLUSION: SMSCs cultured with bFGF promoted the expression of CXCL6. This mechanism may enhance cell migration, proliferation, and chondrogenic differentiation, thereby resulting in superior meniscus regeneration and cartilage preservation.

Modulating pentenoate-functionalized hyaluronic acid hydrogel network properties for meniscal fibrochondrocyte mechanotransduction.

Knee meniscus tears are one of the most common musculoskeletal injuries. While meniscus replacements using allografts or biomaterial-based scaffolds are available, these treatments rarely result in integrated, functional tissue. Understanding mechanotransducive signaling cues that promote a meniscal cell regenerative phenotype is critical to developing therapies that promote tissue regeneration rather than fibrosis after injury. The purpose of this study was to develop a hyaluronic acid (HA) hydrogel system with tunable crosslinked network properties by modulating the degree of substitution (DoS) of reactive-ene groups to investigate mechanotransducive cues received by meniscal fibrochondrocytes (MFCs) from their microenvironment. A thiol-ene step-growth polymerization crosslinking mechanism was employed using pentenoate-functionalized hyaluronic acid (PHA) and dithiothreitol to achieve tunability of the chemical crosslinks and resulting network properties. Increased crosslink density, reduced swelling, and increased compressive modulus (60-1020 kPa) were observed with increasing DoS. Osmotic deswelling effects were apparent in PBS and DMEM+ compared to water; swelling ratios and compressive moduli were decreased in the ionic buffers. Frequency sweep studies showed storage and loss moduli of hydrogels at 1 Hz approach reported meniscus values and showed increasing viscous response with increasing DoS. The degradation rate increased with decreasing DoS. Lastly, modulating PHA hydrogel surface modulus resulted in control of MFC morphology, suggesting relatively soft hydrogels (E = 60 ± 35 kPa) promote more inner meniscus phenotype compared to rigid hydrogels (E = 610 ± 66 kPa). Overall, these results highlight the use of -ene DoS modulation in PHA hydrogels to tune crosslink density and physical properties to understand mechanotransduction mechanisms required to promote meniscus regeneration.

Extracellular vesicles derived from mesenchymal stromal cells mediate endogenous cell growth and migration via the CXCL5 and CXCL6/CXCR2 axes and repair menisci.

BACKGROUND: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are promising candidates for tissue regeneration therapy. However, the therapeutic efficacy of MSC-EVs for meniscus regeneration is uncertain, and the mechanisms underlying MSC-EV-mediated tissue regeneration have not been fully elucidated. The aims of this study were to evaluate the therapeutic efficacy of intra-articular MSC-EV injection in a meniscus defect model and elucidate the mechanism underlying MSC-EV-mediated tissue regeneration via combined bioinformatic analyses. METHODS: MSC-EVs were isolated from human synovial MSC culture supernatants via ultrafiltration. To evaluate the meniscus regeneration ability, MSC-EVs were injected intra-articularly in the mouse meniscus defect model immediately after meniscus resection and weekly thereafter. After 1 and 3 weeks, their knees were excised for histological and immunohistochemical evaluations. To investigate the mechanisms through which MSC-EVs accelerate meniscus regeneration, cell growth, migration, and chondrogenesis assays were performed using treated and untreated chondrocytes and synovial MSCs with or without MSC-EVs. RNA sequencing assessed the gene expression profile of chondrocytes stimulated by MSC-EVs. Antagonists of the human chemokine CXCR2 receptor (SB265610) were used to determine the role of CXCR2 on chondrocyte cell growth and migration induced by MSC-EVs. RESULTS: In the meniscus defect model, MSC-EV injection accelerated meniscus regeneration and normalized the morphology and composition of the repaired tissue. MSC-EVs stimulated chondrocyte and synovial MSC cell growth and migration. RNA sequencing revealed that MSC-EVs induced 168 differentially expressed genes in the chondrocytes and significantly upregulated CXCL5 and CXCL6 in chondrocytes and synovial MSCs. Suppression of CXCL5 and CXCL6 and antagonism of the CXCR2 receptor binding CXCL5 and CXCL6 negated the influence of MSC-EVs on chondrocyte cell growth and migration. CONCLUSIONS: Intra-articular MSC-EV administration repaired meniscus defects and augmented chondrocyte and synovial MSC cell growth and migration. Comprehensive transcriptome/RNA sequencing data confirmed that MSC-EVs upregulated CXCL5 and CXCL6 in chondrocytes and mediated the cell growth and migration of these cells via the CXCR2 axis.

Wnt5a/Platelet-rich plasma synergistically inhibits IL-1ß-induced inflammatory activity through NF-κB signaling pathway and prevents cartilage damage and promotes meniscus regeneration.

Noncanonical Wnt5a is a particularly attractive growth factor to maintain chondrogenesis. Platelet-rich plasma (PRP) is an autologous blood-derived product and a source of bioactive growth factors involved in tissue regeneration. The present study aimed to investigate the effect and inflammation reaction of Wnt5a/PRP on meniscus cells, and evaluate meniscus regeneration and osteoarthritis (OA) prevention by the application of Wnt5a/PRP gel in a rabbit model of massive meniscal defect. In vitro, the proliferation, migration, differentiation, and interleukin-1 beta (IL-1ß) IL-1ß-induced inflammation reaction of meniscus cells treated by Wnt5a and PRP was assessed. In vivo, the anterior half of the medial meniscus of 18 New Zealand rabbits was excised and implanted with PRP gel, Wnt5a/PRP gel or untreated. After 6 and 12 weeks, the regenerated meniscus were evaluated. Wnt5a can promote the migration of meniscus cells. PRP and Wnt5a had synergistic effect in promoting the proliferation and chondrogenic differentiation of meniscus cells. The IL-1ß-induced meniscus cells study showed that PRP and Wnt5a had the anti-inflammatory actions through nuclear factor kB (NF-κB) signaling pathway. PRP and Wnt5a/PRP significantly inhibited the increase of the p-p65/p65 and p-IκB-α/IκB-α ratios. In vivo transplantation of Wnt5a/PRP gel was demonstrated to promote meniscus regeneration, while reducing OA of knee joint. Wnt5a with PRP had the anti-inflammatory activity in an IL-1ß-induced inflammatory model. They can synergistically improve the chondorgenic differentiation of meniscus cells. Wnt5a/PRP gel treatment could potentially be developed into a new method for meniscus regeneration and the prevention of OA.

3D-printed cell-free PCL-MECM scaffold with biomimetic micro-structure and micro-environment to enhance in situ meniscus regeneration.

Despite intensive effort was made to regenerate injured meniscus by cell-free strategies through recruiting endogenous stem/progenitor cells, meniscus regeneration remains a great challenge in clinic. In this study, we found decellularized meniscal extracellular matrix (MECM) preserved native meniscal collagen and glycosaminoglycans which could be a good endogenous regeneration guider for stem cells. Moreover, MECM significantly promoted meniscal fibrochondrocytes viability and proliferation, increased the expression of type II collagen and proteoglycans in vitro. Meanwhile, we designed 3D-printed polycaprolactone (PCL) scaffolds which mimic the circumferential and radial collagen orientation in native meniscus. Taken these two advantages together, a micro-structure and micro-environment dually biomimetic cell-free scaffold was manipulated. This cell-free PCL-MECM scaffold displayed superior biocompatibility and yielded favorable biomechanical capacities closely to native meniscus. Strikingly, neo-menisci were regenerated within PCL-MECM scaffolds which were transplanted into knee joints underwent medial meniscectomy in rabbits and sheep models. Histological staining confirmed neo-menisci showed meniscus-like heterogeneous staining. Mankin scores showed PCL-MECM scaffold could protect articular cartilage well, and knee X-ray examination revealed same results. Knee magnetic resonance imaging (MRI) scanning also showed some neo-menisci in PCL-MECM scaffold group. In conclusion, PCL-MECM scaffold appears to optimize meniscus regeneration. This could represent a promising approach worthy of further investigation in preclinical applications.

A novel kartogenin-platelet-rich plasma gel enhances chondrogenesis of bone marrow mesenchymal stem cells in vitro and promotes wounded meniscus healing in vivo.

BACKGROUND: The meniscus tear is one of the most common knee injuries particularly seen in athletes and aging populations. Subchondral bone sclerosis, irreparable joint damage, and the early onset of osteoarthritis make the injured meniscus heal difficultly. METHODS: The study was performed by in vitro and in vivo experiments. The in vitro experiments were carried out using the bone marrow stem cells (BMSCs) isolated from the rabbits, and the stemness of the BMSCs was tested by immunostaining. The BMSCs positively expressed stem cell markers were cultured with various concentrations of kartogenin (KGN) for 2 weeks. The chondrogenesis of BMSCs induced by KGN was examined by histochemical staining and quantitative RT-PCR. The in vivo experiments were completed by a rabbit model. Three holes were created in each meniscus by a biopsy punch. The rabbits were treated with four different conditions in each group. Group 1 was treated with 20 µl of saline (saline); group 2 was treated with 5 µl of 100 µM KGN and 15 µl saline (KGN); group 3 was treated with 5 µl of 100 µM KGN, 5 µl of 10,000 U/ ml thrombin, and 10 µl of PRP (KGN+PRP); group 4 was treated with 10,000 BMSCs in 10 µl of PRP, 5 µl of saline solution, and 5 µl of 10,000 U/ml thrombin (PRP+BMSC); group 5 was treated with 10,000 BMSCs in 10 µl of PRP, 5 µl of 100 µM KGN, and 5 µl of 10,000 U/ml thrombin (KGN+PRP+BMSC). The menisci were collected at day 90 post-surgery for gross inspection and histochemical analysis. RESULTS: The histochemical staining showed that KGN induced chondrogenesis of BMSCs in a concentration-dependent manner. The RT-PCR results indicated that chondrocyte-related genes were also increased in the BMSCs cultured with KGN in a dose-dependent manner. The in vivo results showed that large unhealed wound areas were still found in the wounds treated with saline and KGN groups. The wounds treated with BMSCs-containing PRP gel healed much faster than the wounds treated without BMSCs. Furthermore, the wounds treated with BMSCs-containing KGN-PRP gel have healed completely and formed more cartilage-like tissues than the wounds treated with BMSCs-containing PRP gel. CONCLUSIONS: BMSCs could be differentiated into chondrocytes when they were cultured with KGN-PRP gel in vitro and formed more cartilage-like tissues in the wounded rabbit meniscus when the wounds were treated with BMSCs-containing KGN-PRP gel. The results indicated that the BMSCs-containing KGN-PRP gel is a good substitute for injured meniscus repair and regeneration.

Bone Marrow Aspirate Concentrate for the Treatment of Avascular Meniscus Tears in a One-Step Procedure-Evaluation of an In Vivo Model.

Avascular meniscus tears show poor intrinsic regenerative potential. Thus, lesions within this area predispose the patient to developing knee osteoarthritis. Current research focuses on regenerative approaches using growth factors or mesenchymal stem cells (MSCs) to enhance healing capacity within the avascular meniscus zone. The use of MSCs especially as progenitor cells and a source of growth factors has shown promising results. However, present studies use bone-marrow-derived BMSCs in a two-step procedure, which is limiting the transfer in clinical praxis. So, the aim of this study was to evaluate a one-step procedure using bone marrow aspirate concentrate (BMAC), containing BMSCs, for inducing the regeneration of avascular meniscus lesions. Longitudinal meniscus tears of 4 mm in size of the lateral New Zealand White rabbit meniscus were treated with clotted autologous PRP (platelet-rich plasma) or BMAC and a meniscus suture or a meniscus suture alone. Menisci were harvested at 6 and 12 weeks after initial surgery. Macroscopical and histological evaluation was performed according to an established Meniscus Scoring System. BMAC significantly enhanced regeneration of the meniscus lesions in a time-dependent manner and in comparison to the PRP and control groups, where no healing could be observed. Treatment of avascular meniscus lesions with BMAC and meniscus suturing seems to be a promising approach to promote meniscus regeneration in the avascular zone using a one-step procedure.

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus.

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60 mW/cm2 intensity for 20 min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4 weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

Enzyme-mediated tissue adhesive hydrogels for meniscus repair.

Meniscus tissues have limited regenerative capacity once damaged. The treatment options for the meniscus tissue regeneration have been limited to arthroscopic meniscectomy or surgical interventions. The injectable hydrogels based system would provide an alternative to the conventional meniscus therapy by providing a minimally invasive treatment alternative. Here we utilized enzyme-based approaches to fabricate tissue adhesive hydrogels for meniscus repair. Tyramine (TA) conjugated hyaluronic acid (TA-HA) and gelatin are susceptible to tyrosinase (TYR)-mediated crosslinking in vitro and in vivo. Importantly, mechanical properties and degradation kinetics are modulated by the TA substitution and TYR concentrations. In addition, TYR -mediated crosslinking displayed tissue-adhesive properties. Furthermore, fibrochondrocyte-laden and TYR-crosslinked hydrogels demonstrated strong biocompatibility and resulted in enhancement of cartilage-specific gene expression and matrix synthesis. Overall, this represents a potential application of enzyme-mediated crosslinking hydrogels for meniscus tissue engineering.

Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

BACKGROUND: Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis. METHODS: A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis. RESULTS: After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05). CONCLUSIONS: The regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

A Novel Approach for Meniscal Regeneration Using Kartogenin-Treated Autologous Tendon Graft.

BACKGROUND: The meniscus is one of the most commonly injured parts of the body, and meniscal healing is difficult. HYPOTHESIS: Kartogenin (KGN) induces tendon stem cells (TSCs) to differentiate into cartilage cells in vitro and form meniscus-like tissue in vivo. A damaged meniscus can be replaced with a KGN-treated autologous tendon graft. STUDY DESIGN: Controlled laboratory study. METHODS: In the in vitro experiments, TSCs were isolated from rabbit patellar tendons and cultured with various concentrations of KGN, from 0 to 1000 µM. The effect of KGN on the chondrogenesis of TSCs in vitro was investigated by histochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The in vivo experiments were carried out on 6 New Zealand White rabbits by removing a meniscus from the rabbit knee and implanting an autologous tendon graft treated with KGN or saline. The meniscus formation in vivo was examined by histological analysis and immune staining. RESULTS: The proliferation of TSCs was promoted by KGN in a concentration-dependent manner. Both histochemical staining and qRT-PCR showed that the chondrogenic differentiation of TSCs was increased with KGN concentration. After 3 months of implantation, the tendon graft treated with KGN formed a meniscus-like tissue with a white and glistening appearance, while the saline-treated tendon graft retained tendon-like tissue and appeared yellowish and unhealthy. Histochemical staining showed that after 3 months of implantation, the KGN-treated tendon graft had a structure similar to that of normal meniscus. Many cartilage-like cells and fibrocartilage-like tissues were found in the KGN-treated tendon graft. However, no cartilage-like cells were found in the saline-treated tendon graft after 3 months of implantation. Furthermore, the KGN-treated tendon graft was positively stained by both anti-collagen type I and type II antibodies, but the saline-treated tendon graft was not stained by collagen type II. CONCLUSION: The findings indicated that KGN can induce the differentiation of TSCs into cartilage-like cells in vitro and in vivo. The results suggest that KGN-treated tendon graft may be a good substitute for meniscal repair and regeneration. CLINICAL RELEVANCE: This study revealed the direct effects of KGN on the chondrogenic differentiation of TSCs in vitro and in vivo. A KGN-treated autologous tendon graft induced formation of a meniscus-like tissue in vivo. This study provides a new cartilage regenerating technology for the treatment of damaged meniscus.

Do cell based tissue engineering products for meniscus regeneration influence vascularization?

BACKGROUND: Meniscus regeneration is observed within the peripheral, vascularized zone but decreases in the inner two thirds alongside the vascularization. Within this avascular area, cell-based tissue-engineering-approaches appear to be a promising strategy for the treatment of meniscal defects. OBJECTIVE: Evaluation of the angiogenic potential of cell-based tissue-engineering-products for meniscus healing. METHODS: Evaluation of angiogenesis induced by rabbit meniscus-pellets, meniscus-cells (MC) or mesenchymal stem-cells (MSC) in cell-based tissue-engineering-products within a rabbit meniscus-ring was performed using a transparent dorsal skin fold chamber in nude mice. Observations were undertaken during a 14 days period. Cell preconditioning differed between experimental groups. Immunohistochemical analysis of the regenerated tissue in the meniscus-ring induced by cell loaded composite scaffolds for differentiation and anti-angiogenic factors were performed. RESULTS: Meniscus-pellets and MSC-/MC-based tissue-engineering-products induced angiogenesis. An accelerated vascularization was detected in the group of meniscus-pellets derived from the vascularized zone compared to avascular meniscus-pellets. In terms of cell-based tissue-engineering-products, chondrogenic preconditioning resulted in significantly increased vessel growth. MSC-constructs showed an accelerated angiogenesis. Immunohistochemical evaluation showed a progressive differentiation and lower content for anti-angiogenic endostatin in the precultured group. CONCLUSIONS: Preconditioning of MC-/MSC-based tissue-engineering-products is a promising tool to influence the angiogenic potential of tissue-engineering-products and to adapt these properties according to the aimed tissue qualities.

Poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering.

Regenerative engineering converges tissue engineering, advanced materials science, stem cell science, and developmental biology to regenerate complex tissues such as whole limbs. Regenerative engineering scaffolds provide mechanical support and nanoscale control over architecture, topography, and biochemical cues to influence cellular outcome. In this regard, poly (lactic acid) (PLA)-based biomaterials may be considered as a gold standard for many orthopaedic regenerative engineering applications because of their versatility in fabrication, biodegradability, and compatibility with biomolecules and cells. Here we discuss recent developments in PLA-based biomaterials with respect to processability and current applications in the clinical and research settings for bone, ligament, meniscus, and cartilage regeneration.

An injectable extracellular matrix derived hydrogel for meniscus repair and regeneration.

Tissue-derived extracellular matrix (ECM) biomaterials to regenerate the meniscus have gained increasing attention in treating meniscus injuries and diseases, particularly for aged persons and athletes. However, ECM scaffold has poor cell infiltration and can only be implanted using surgical procedures. To overcome these limitations, we developed an injectable ECM hydrogel material from porcine meniscus via modified decellularization and enzymatic digestion. This meniscus-derived ECM hydrogel exhibited a fibrous morphology with tunable compression and initial modulus. It had a good injectability evidenced by syringe injection into mouse subcutaneous tissue. The hydrogel showed good cellular compatibility by promoting the growth of both bovine chondrocytes and mouse 3T3 fibroblasts encapsulated in the hydrogel for 2 weeks. It also promoted cell infiltration as shown in both in vitro cell culture and in vivo mouse subcutaneous implantation. The in vivo study revealed that the ECM hydrogel possessed good tissue compatibility after 7 days of implantation. The results support the great potential of the newly produced injectable meniscus-derived ECM hydrogel specifically for meniscus repair and regeneration.

Meniscus regeneration by syngeneic, minor mismatched, and major mismatched transplantation of synovial mesenchymal stem cells in a rat model.

We compared the effect of syngeneic and allogeneic transplantation of synovial mesenchymal stem cells (MSCs) for meniscus regeneration in a rat model. Synovium was harvested from the knee joints of three strains of rats. The anterior half of the medial meniscus in both knees of F344 rats was removed and 5 million synovial MSCs derived from F344 (syngeneic transplantation), Lewis (minor mismatched transplantation), and ACI (major mismatched transplantation) were injected into the knee of the F344 rats. At 4 weeks, the area of the regenerated meniscus in the F344 group was significantly larger than that in the ACI group. Histological score was significantly better in the F344 and Lewis groups than in the ACI group at 8 weeks. DiI labeled cells could be observed in the knee joint in the F344 group, but were hardly detected in the ACI group at 1 week. The number of macrophages and CD8 T cells at synovium around the meniscus defect was significantly lower in the F344 group than in the ACI group at 1 week. Syngeneic and minor mismatched transplantation of synovial MSCs promoted meniscus regeneration better than major mismatched transplantation in a rat meniscectmized model.

PLDLA/PCL-T Scaffold for Meniscus Tissue Engineering.

The inability of the avascular region of the meniscus to regenerate has led to the use of tissue engineering to treat meniscal injuries. The aim of this study was to evaluate the ability of fibrochondrocytes preseeded on PLDLA/PCL-T [poly(L-co-D,L-lactic acid)/poly(caprolactone-triol)] scaffolds to stimulate regeneration of the whole meniscus. Porous PLDLA/PCL-T (90/10) scaffolds were obtained by solvent casting and particulate leaching. Compressive modulus of 9.5±1.0 MPa and maximum stress of 4.7±0.9 MPa were evaluated. Fibrochondrocytes from rabbit menisci were isolated, seeded directly on the scaffolds, and cultured for 21 days. New Zealand rabbits underwent total meniscectomy, after which implants consisting of cell-free scaffolds or cell-seeded scaffolds were introduced into the medial knee meniscus; the negative control group consisted of rabbits that received no implant. Macroscopic and histological evaluations of the neomeniscus were performed 12 and 24 weeks after implantation. The polymer scaffold implants adapted well to surrounding tissues, without apparent rejection, infection, or chronic inflammatory response. Fibrocartilaginous tissue with mature collagen fibers was observed predominantly in implants with seeded scaffolds compared to cell-free implants after 24 weeks. Similar results were not observed in the control group. Articular cartilage was preserved in the polymeric implants and showed higher chondrocyte cell number than the control group. These findings show that the PLDLA/PCL-T 90/10 scaffold has potential for orthopedic applications since this material allowed the formation of fibrocartilaginous tissue, a structure of crucial importance for repairing injuries to joints, including replacement of the meniscus and the protection of articular cartilage from degeneration.

Matrix formation is enhanced in co-cultures of human meniscus cells with bone marrow stromal cells.

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co-cultured in vitro in three-dimensional (3D) pellet culture at three different cell-cell ratios for 3 weeks under normal (21% O2 ) or low (3% O2 ) oxygen tension in the presence of serum-free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT-PCR. Co-cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3-1.7-fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O2 . GAG matrix formation was also enhanced (1.3-1.6-fold) under 3% O2 culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG-rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co-cultured pellets. However, SOX9 and HIF-1α mRNA expression were not significantly modulated by co-culture. Co-culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co-delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects.