Dopamine-functionalized bioinspired pre-chondrogenic hydrogel for cell-free cartilage regeneration.

Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.

Hyaluronic acid (HA) based hydrogels are one of most functional natural biomaterials in the field of cartilage tissue engineering (CTE). Even with the promising advantages of HA hydrogels, the complicated mechanical properties of the native cartilage have not been realized, and fabricating HA hydrogels with excellent mechanical properties to make them practical in CTE still remains a current challenge. Here, a strategy that integrates hydrogels and nanomaterials is shown to form a HA hydrogel with sufficient mechanical loading for cartilage tissue production and recombination. Cellulose nanofibrils (CNFs) are promising nanomaterial candidates as they possess high mechanical strength and excellent biocompatibility. In this study, we developed methacrylate-functionalized CNFs that are able to photo-crosslink with methacrylated HA to fabricate HA/CNF nanocomposite hydrogels. The present composite hydrogels with a compressive modulus of 0.46 ± 0.05 MPa showed adequate compressive strength (0.198 ± 0.009 MPa) and restorability, which can be expected to employ as a stress-bearing tissue such as articular cartilage. Besides, this nanocomposite hydrogel could provide a good microenvironment for bone marrow mesenchymal stem cell proliferation, as well as chondrogenic differentiation, and exhibit prominent repair effect in the full-thickness cartilage defect model of SD rats. These results suggest that the HA/CNF nanocomposite hydrogel creates a new possibility for fabricating a scaffold in CTE.

Injectable double-crosslinked hydrogels with kartogenin-conjugated polyurethane nano-particles and transforming growth factor β3 for in-situ cartilage regeneration.

Articular cartilage has poor capability of regeneration due to the avascular surrounding and low metabolic activity. Kartogenin (KGN), an emerging nonprotein heterocyclic compound, was screened to stimulate chondrogenic differentiation of bone mesenchymal stem cells (BMSCs). Coculturing BMSCs and chondrocytes was reported to overcome the shortcomings of forming fibroblastic and hypertrophic cartilages. In this study, KGN was incorporated into the Col-Tgel hydrogel to form a Gel/Cell/KGN complex, which fabricated an appropriate microenvironment for effective cartilage regeneration of BMSCs and/or chondrocytes. The complexes that incorporated KGN, BMSCs, and chondrocytes achieved higher lubricin expression and extracellular matrix production, such as characteristic glycosaminoglycans (GAGs) and collagen type II (COL II), compared to the monocultures of BMSCs or chondrocytes in vitro. The complexes compounding KGN, BMSCs, and chondrocytes (at an optimal ratio in the in vitro experiment) were transplanted into rat models to evaluate the repair effects. Our results suggested that the interaction between BMSCs and chondrocytes can substitute the use of growth factors to some degree and indicated the role of KGN in chondrogenesis induction. Besides, it is the first time (to our knowledge) that the expression of lubricin was found to be delayed in the coculture of mixed cells comparing with GAGs and COL II, which could be significant in cartilage tissue engineering.

Objective To investigate the feasibility of bone marrow mesenchymal stem cells (BMSCs) combined with type Ⅱ collagen- hyaluronic acid- oxidized chondroitin sulfate (Col Ⅱ-HA-OCS) biomimetic hydrogel to repair articular cartilage defect in porcine and the role of the transplanted cells played in the process of cartilage repair. Methods A articular cartilage defect model which remaining cartilage calcified zone was created in the knee of Bama minipigs, the autologous BMSCs was used as seeds for transplantation and was labeled by the 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFDA SE). Animals were randomly divided into three groups: Group A (blank group) was left untreated, group B (cell-free biomimetic hydrogel group) was filled with biomimetic hydrogel and group C (BMSCs combined with biomimetic hydrogel group) was filled with the CFDA SE labeled autologous BMSCs combined with biomimetic hydrogel. One month after the operation, BrdU labeled liquid was injected intravenously into the animals 24 h and 48 h before specimens were taken from the executed animals. Partial cartilage repaired tissue in Group C was taken, cryosectioned and stained with DAPI and BrdU immunofluorescence. Confocal laser scanning microscope was used to observe and count the cells. Specimens of the three groups were analyzed through gross observation and histological staining, and scored according to the international cartilage repair society (ICRS) gross morphological score and ICRS histological score. Results Laser scanning confocal microscopy showed (97.3±2.6)% of the cells were derived from the implanted BMSCs in repaired tissue and that the ratio of these cells with proliferative capacity was (76.6±2.5)%. Gross observation suggested most of the cartilage defect areas in Group C were filled with ivory tissue, but those in Group A and B were still obvious depression. Histological staining showed the cartilage defect areas in Group C were filled with cartilage like tissue, which was well integrated with the surrounding normal cartilage, presented a few cartilage lacunas could be seen, and had contents of Col Ⅱ and glycosaminoglycan similar with the adjacent normal cartilage. There was almost no filler in the defect area in Group A. There was little fibrous tissue in the defect area in Group B. ICRS gross score was (8.3±1.0)points in Group C, higher than that in Group A [(0.5±0.6)points] and Group B [(2.3±0.5)points] (P<0.05). ICRS histological score was (10.3±2.4)points in Group C, higher than that in Group A [(0.5±0.6)points] and Group B [(4.5±1.0)points] (P<0.05). Conclusions BMSCs combined with Col Ⅱ-HA-OCS biomimetic hydrogel for repairing porcine articular cartilage defects can achieve satisfactory results. Implanted BMSCs are the main component of the cell composition in the repaired tissue and gradually differentiated into chondrocytes. Key words: Cartilage; Mesenchymal stem cells; Collagen type Ⅱ; Bone regeneration

Cartilage defects are frequently caused by trauma, illness and degradation of the cartilage. If these defects are not sufficiently treated, the joints will degrade irreversibly, possibly resulting in disability. Articular cartilage lacks blood vessels and nerves and is unable to regenerate itself, so the repair of cartilage defects is extremely challenging in clinical treatment. Tissue engineering technology is an emerging technology in cartilage repair and cartilage regeneration. 3D-printed hydrogels show great potential in cartilage tissue engineering for the fabrication of 3D cell culture scaffolds to mimic extracellular matrix. In this study, we construct a 3D-printed hydrogel loaded with nanoparticles by electrostatic interaction and photo cross-linking for the regeneration of cartilage, which has adaptable and drug-continuous release behavior. A photopolymerizable bioink was prepared using recombinant collagen, chitosan, nanoclay Laponite-XLG and nanoparticles loaded with Kartogenin (KGN). This bioink was added with KGN, a small molecule drug that promotes cartilage differentiation, and as a result, the 3D-printed CF/CM/3%LAP/KGN scaffolds obtained by extrusion printing is expected to be used for cartilage repair. It was shown that the 3D-printed scaffolds had good cytocompatibility for human bone marrow mesenchymal stem cells (hBMSCs) and exhibited excellent antimicrobial properties, the continuous release of KGN in the scaffold induced the hBMSCs differentiation into chondrocytes, which significantly enhanced the expression of collagen II and glycosaminoglycan. In vivo studies have shown that implantation of KGN-loaded scaffolds into cartilage-injured tissues promoted cartilage tissue regeneration. This study demonstrated that 3D-printed CF/CM/3%LAP/KGN scaffolds can be used for cartilage repair, which is expected to lead to new healing opportunities for cartilage injury-based diseases.

In cartilage tissue engineering using stem cells, it is important to stimulate proliferation and control the differentiation of stem cells to specific lineages. Here we reported a combined technique for articular cartilage repair, consisting of bone marrow mesenchymal stem cells (BMMSCs) transfected with connective tissue growth factor (CTGF) gene and NaOH-treated poly(lactic-co-glycolic) acid (PLGA) scaffolds. In the present study, BMMSCs or CTGF-modified BMMSCs seeded on PLGA or NaOH-treated PLGA scaffolds were incubated in vitro and NaOH-treated PLGA significantly stimulated proliferation of BMMSCs, while CTGF gene transfer promoted chondrogenic differentiation. The effects of the composite on the repair of cartilage defects were evaluated in rabbit knee joints in vivo. Full-thickness cartilage defects (diameter: 5 mm; depth: 3 mm) were created unilaterally in the patellar groove. Defects were either left empty (n = 18) or implanted with BMMSCs/PLGA (n = 18), BMMSCs/NaOH-treated PLGA (n = 18), or CTGF-modified BMMSCs/NaOH-treated PLGA (n = 18). The defect area was examined grossly, histologically, and mechanically at 6, 12, and 24 weeks postoperatively. Implanted cells were tracked using adeno-LacZ labeling at 6 weeks after implantation. Overall, the CTGF-modified BMMSCs/NaOH-treated PLGA group showed successful hyaline-like cartilage regeneration similar to normal cartilage, which was superior to the other groups using gross examination, qualitative and quantitative histology, and mechanical assessment. The in vivo viability of the implanted cells was demonstrated by their retention for 6 weeks after implantation. These findings suggested that a combination of CTGF-modified BMMSCs and NaOH-treated PLGA may be an alternative treatment for large osteochondral defects in high-loading sites.

Recent studies suggest the presence of cell adhesion motifs found in structural proteins can inhibit chondrogenesis. In this context, the current study aims to determine if a polyethylene glycol (PEG)-modified fibrinogen matrix could support better chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSC) based on steric interference of adhesion, when compared to a natural fibrin matrix. Hydrogels used as substrates for two-dimensional (2D) BM-MSC cultures under chondrogenic conditions were made from cross-linked PEG-fibrinogen (PF) and compared to thrombin-activated fibrin. Cell morphology, protein expression, DNA and sulfated proteoglycan (GAG) content were correlated to substrate properties such as stiffness and adhesiveness. Cell aggregation and chondrogenic markers, including collagen II and aggrecan, were observed on all PF substrates but not on fibrin. Shielding fibrinogen’s adhesion domains and increasing stiffness of the material are likely contributing factors that cause the BM-MSCs to display a more chondrogenic phenotype. One composition of PF corresponding to GelrinC™—a product cleared in the EU for cartilage repair—was found to be optimal for supporting chondrogenic differentiation of BM-MSC while minimizing hypertrophy (collagen X). These findings suggest that semi-synthetic biomaterials based on ECM proteins can be designed to favourably affect BM-MSC towards repair processes involving chondrogenesis.

A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-β1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro chondrogenic potency of TGF-β1 and KGN using a high resolution micropellet model system. Micropellets were cultured for 7–14 days in medium supplemented with TGF-β1, KGN, or both TGF-β1 + KGN. Following 14 days of induction, micropellets exposed to TGF-β1 alone or TGF-β1 + KGN in combination were larger and produced more glycosominoglycan (GAG) than KGN-only cultures. When TGF-β1 + KGN was used, GAG quantities were similar or slightly greater than the TGF-β1-only cultures, depending on the BMSC donor. BMSC micropellet cultures supplemented with KGN alone contracted in size over the culture period and produced minimal GAG. Indicators of hypertrophy were not mitigated in TGF-β1 + KGN cultures, suggesting that KGN does not obstruct BMSC hypertrophy. KGN appears to have weak chondrogenic potency in human BMSC cultures relative to TGF-β1, does not obstruct hypertrophy, and may not be a viable alternative to growth factors in cartilage tissue engineering.

Objective To investigate the effect of hyaluronan (HA) on engineered cartilage formation.Methods Human mesenchymal stem cell (hBMSCs) were isolated by density gradient centrifugation and were seeded onto tricalcium phosphate-collagen (TCP-COL) scaffolds.hBMSCs were cultured on the scaffold for 2 weeks in two groups:(1) control group ; (2) HA group (control group culture medium with 100 mg/L HA).The effect of hyaluronan on chondrogenesis was assessed by hematoxylin-eosin (HE) staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,Real-time polymerase chain reaction (RT-PCR) and glycosaminoglycans (GAG) quantification assay.Results The data of hematoxylin-eosin (HE) staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,RT-PCR [Collagen Ⅱ (COL2) (P ＜ 0.01),SRY-related high mobility group-box gene9 (SOX9) (P ＜ 0.01),Aggrecan (P ＜ 0.05)] (8.954 ± 2.612/1.062 ± 0.459,3.512 ± 0.638/1.111 ± 0.595 and 4.588 ± 1.964/1.042 ± 0.335,respectively) and GAG quantification assay (21.741 ± 2.633/16.717 ± 0.595) (P ＜ 0.05) shows HA (100 g/L) could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffold.In addition,in the HA group,a significant up-regulation of COL1 (P＜0.05) (3.258 ±1.038/1.127 ±0.678) andCOL10 (P＜0.05) (5.961 ±2.598/1.080 ±0.458) gene were detected compared with the control group,indicating that HA may also promote robust maturation of tissue-engineered cartilage.Conclusion HA not only could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffolds,but also could promote maturation of the tissue-engineered cartilage. Key words: Hyaluronic acid; Bone marrow mesenchymal stem cells; Tissue-engineering cartilage

This study focuses on the development of injectable hydrogels to mimic the cartilage microenvironment using hyaluronic acid (HA) derivatives as starting materials. Cysteine-inserted Tobacco mosaic virus (TMV) mutants (TMV1cys) could be cross-linked to methacrylated hyaluronic acid (MeHA) polymers by thiol-ene "click" chemistry and form hydrogels under physiological condition. The resulting hydrogels could promote in vitro chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) significantly higher than that in the TMV-free HA hydrogels by upregulating bone morphogenetic protein-2 (BMP-2) expression and enhancing collagen accumulation.

Recently, cartilage tissue engineering (CTE) attracts increasing attention in cartilage defect repair. In this work, kartogenin (KGN), an emerging chondroinductive nonprotein small molecule, was incorporated into a thermogel of poly(L-lactide-co-glycolide)-poly(ethylene glycol)-poly(L-lactide-co-glycolide) (PLGA-PEG-PLGA) to fabricate an appropriate microenvironment of bone marrow mesenchymal stem cells (BMSCs) for effective cartilage regeneration. More integrative and smoother repaired articular surface, more abundant characteristic glycosaminoglycans (GAGs) and collagen II (COL II), and less degeneration of normal cartilage were obtained in the KGN and BMSCs coloaded thermogel group in vivo. In conclusion, the KGN-loaded PLGA-PEG-PLGA thermogel can be utilized as an alternative support for BMSCs to regenerate damaged cartilage in vivo.

Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness.

P-glycoprotein (P-gp) is an adenosine-5-triphosphate Binding Cassettes B 1 (ABCB1) transporter that exports various substrates on cellular membrane. Surface expression of P-gp was decreased during chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSCs). We examined the role of P-gp in extracellular matrix deposition during chondrogenesis of human BM-MSCs. BM-MSCs were isolated from 16 volunteers after informed consent and incubated for 28 days using three-dimensional culture methods in chondrogenic medium with and without P-gp inhibitor (verapamil, 10 μmol/L). Hematoxylin and eosin staining revealed a cartilaginous structure with chondrogenic cells in the lacunae after 2 weeks of culture. Total glycosaminoglycan (GAG) content was increased and rose during pellet culture. Hyaluronan (HA) content of the culture medium decreased with P-gp inhibitor. Type II collagen deposition decreased with P-gp inhibitor. Inhibition of P-gp facilitated GAG accumulation via HA export inhibition during chondrogenic differentiation of human BM-MSCs. Modulation of P-gp expression during chondrogenesis would be a possible therapeutic approach for articular cartilage regeneration.

Injectable acellular matrix microgel assembly with stem cell recruitment and chondrogenic differentiation functions promotes microfracture-based articular cartilage regeneration