Xingdong Zhang

Chondrogenic differentiation of mesenchymal stem cells induced by collagen-based hydrogel: an in vivo study.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) relies on inductive media of chondrogenic environment. With proper design, a cellular microenvironment mimicking chondrogenic environment might be created to induce chondrogenesis of MSCs. In this study, bone marrow mesenchymal cells (BMSCs) were encapsulated in collagen-based hydrogel, and then enclosed in diffusion-chambers which allow the body fluid to permeate and preclude the host cells to invade. Then, the chamber with the hydrogel-BMSCs composite was implanted in the back of rabbits subcutaneously. The specimens in the chamber were harvested for histological, immunohistochemical, and RT-PCR analyses after 8 weeks. The results showed that cells with the characteristic of chondrocytes were homogenously distributed and the extracellular matrix (ECM) of cartilage has been secreted, indicating the chondrogenic differentiation of BMSCs. As control, nothing was obtained with only BMSCs. Moreover, the expression of collagen type II, indicator of cartilage ECM, was less in tissues with collagen-alginate-hydrogel (CAH) than that with collagen-hydrogel (CH). The results showed that both CH and CAH may induce the chondrogenesis and the induction is materials dependent. From in vitro experiments, TGF-beta is a necessary signal molecule for chondrogenesis, and it was suggested that the material may take in vivo growth factors to trigger chondrogenesis. From the studies, the chondrogenic induction of the hydrogel may be ascribed to that the hydrogel may provide a suitable environment and aggregate the signal molecule for chondrogenesis in vivo. The results would lend valuable reference in clinical for selection of appropriate scaffold for cartilage repair.

In Vivo Cartilage Engineering with Collagen Hydrogel and Allogenous Chondrocytes After Diffusion Chamber Implantation in Immunocompetent Host

In vivo cartilage reconstruction at an ectopic site was not successful in immunocompetent animals, possibly because of immunoreaction and the failure of material design. A diffusion chamber, which has been predominantly adopted to study cell differentiation, was effective in preventing host immune rejection, host cell invasion, and vascular invasion. In this study, we proposed to regenerate ectopic cartilage tissue in rabbits by implanting a diffusion-chamber system subcutaneously for 8 weeks. Inside the chamber, biomimetic scaffolds loaded with allogenous chondrocytes from newborn rabbits were enclosed. Tissue with characteristics of cartilage was formed inside the chamber with collagen gel as a scaffold, which was demonstrated using histological, immunohistochemical, and reverse transcriptase polymerase chain reaction assays. In contrast, for implant without diffusion chamber, vascular invasion was observed and results showed much less expression of cartilage extracellular matrix (ECM). Collagen type I hydrogel and sponge were compared as scaffolds. No cartilage tissue was found in the collagen sponge inside the chamber, presumably because of the different cell-seeding characteristics of gel. In addition, allogenous chondrocytes were adopted as a cell resource and were proved viable for the regeneration of cartilage tissue in this model. The results revealed that the diffusion chamber and scaffold design are both important in providing a more favorable biomimetic microenvironment for the formation of cartilage in vivo at an ectopic site, even with allogenous cells. Moreover, preliminary repair of a cartilage defect using the engineered tissue for 4 weeks showed the growth of new cartilage, obtaining a satisfactory interface with the original cartilage inside the defect. The model of engineering cartilage in vivo was proven to be useful. This study is the preliminary exploration for the reconstruction of ectopic cartilage in an immunocompetent host to be applied for cartilage repair. It may provide a valuable reference for the clinical application of cartilage repair.

Computer simulation of biomolecule–biomaterial interactions at surfaces and interfaces

Biomaterial surfaces and interfaces are intrinsically complicated systems because they involve biomolecules, implanted biomaterials, and complex biological environments. It is difficult to understand the interaction mechanism between biomaterials and biomolecules through conventional experimental methods. Computer simulation is an effective way to study the interaction mechanism at the atomic and molecular levels. In this review, we summarized the recent studies on the interaction behaviors of biomolecules with three types of the most widely used biomaterials: hydroxyapatite (HA), titanium oxide (TiO2), and graphene(G)/graphene oxide(GO). The effects of crystal forms, crystallographic planes, surface defects, doping atoms, and water environments on biomolecules adsorption are discussed in detail. This review provides valuable theoretical guidance for biomaterial designing and surface modification.

Evaluation of novel in situ synthesized nano-hydroxyapatite/collagen/alginate hydrogels for osteochondral tissue engineering

Collagen hydrogel has been widely used for osteochondral repair, but its mechanical properties cannot meet the requirements of clinical application. Previous studies have shown that the addition of either polysaccharide or inorganic particles could reinforce the polymer matrix. However, their synergic effects on collagen-based hydrogel have seldom been studied, and the potential application of triple-phased composite gel in osteochondral regeneration has not been reported. In this study, nano-hydroxyapatite (nano-HA) reinforced collagen-alginate hydrogel (nHCA) was prepared by the in situ synthesis of nano-HA in collagen gel followed by the addition of alginate and Ca(2+). The properties of triple-phased nHCA hydrogel were studied and compared with pure collagen and biphasic gels, and the triple-phased composite of collagen-alginate-HA gels showed a superiority in not only mechanical but also biological features, as evidenced by the enhanced tensile and compressive modulus, higher cell viability, faster cell proliferation and upregulated hyaline cartilage markers. In addition, it was found that the synthesis process could also affect the properties of the triple-phased composite, compared to blend-mixing HCA. The in situ-synthesized nHCA hydrogel showed an enhanced tensile modulus, as well as enhanced biological features compared with HCA. Our study demonstrated that the nHCA composite hydrogel holds promise in osteochondral regeneration. The addition of alginate and nano-HA contribute to the increase in both mechanical and biological properties. This study may provide a valuable reference for the design of an appropriate composite scaffold for osteochondral tissue engineering.

Controlled release of 9-nitro-20(S)- camptothecin from methoxy poly(ethylene glycol)-poly(D,L-lactide) micelles

9-nitro-20(S)-camptothecin (9-NC) is a potent topoisomerase-I inhibitor, and it was applied for clinical trials in cancer treatment. However, the applications of 9-NC were limited by its poor solubility and instability. In order to overcome these disadvantages, 9-NC was encapsulated in amphiphilic copolymer micelles composed of methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA, PELA). Three diblock copolymers with different PDLLA chain lengths were synthesized. The critical micelle concentration was varied from 10(-4) g L(-1) to 10(-2) g L(-1). The 9-NC loaded micelles were nanospheres with diameters ranging from 30 nm to 60 nm. The relationship between the composition of copolymers and the drug loading content was discussed. The encapsulation of micelles improved the solubility of 9-NC greatly. The solubility of 9-NC in micelle M1 was about 250 times higher than that of 9-NC in a phosphate buffer solution (PBS). The stability of 9-NC in micelles was also promoted. After being incubated in PBS for 160 min, 80% of 9-NC in micelles existed as an active lactone form, while 85% of 9-NC in PBS were transferred to an inactive carboxylate salt form. The release experiments were carried out in PBS and the results showed that the release processes were controllable.

In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects

In vitro cultured autologous mesenchymal stem cells (MSCs) within passage 5 have been approved for clinical application in stem cell-based treatment of cartilage defects. However, their chondrogenic potential has not yet been questioned or verified. In this study, the chondrogenic potential of bone marrow MSCs at passage 3 (P3 BMSCs) was investigated both in cartilage repair and in vitro, with freshly isolated bone marrow mononuclear cells (BMMNCs) as controls. The results showed that P3 BMSCs were inferior to BMMNCs not only in their chondrogenic differentiation ability but also as candidates for long-term repair of cartilage defects. Compared with BMMNCs, P3 BMSCs presented a decay in telomerase activity and a change in chromosomal morphology with potential anomalous karyotypes, indicating senescence. In addition, interindividual variability in P3 BMSCs is much higher than in BMMNCs, demonstrating genomic instability. Interestingly, remarkable downregulation in cell cycle, DNA replication and mismatch repair (MMR) pathways as well as in multiple genes associated with telomerase activity and chromosomal stability were found in P3 BMSCs. This result indicates that telomerase and chromosome anomalies might originate from expansion, leading to impaired stemness and pluripotency of stem cells. In vitro culture and expansion are not recommended for cell-based therapy, and fresh BMMNCs are the first choice.

Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells

SOX9 plays an important role in mesenchymal condensations during the early development of embryonic skeletons. However, its function in the chondrogenic differentiation of adult mesenchymal stem cells (MSCs) has not been fully investigated because SOX9 RNA interference in adult MSCs has seldom been studied. This study used SOX9 gene as the target gene and the quantum dot (QD)-based nanomaterial QD-NH2 (ZnS shell and poly-ethylene glycol (PEG) coating) with a fluorescent tracer function as the gene carrier to transfect siSOX9 into MSCs after sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation in vitro and in vivo. The results showed that QD-SMCC could effectively bind and deliver siRNAs into the MSCs, followed by efficient siRNA escape from the endosomes. The siRNAs released from QD-SMCC retained their structural integrity and could effectively inhibit the targeted gene expression, leading to reduced chondrogenic differentiation of MSCs and delayed cartilage repair. QDs were excreted from living cells instead of dead cells, and the ZnS shell and PEG coating layer greatly reduced the cytotoxicity of the QDs. The transfection efficiency of QD-SMCC was superior to that of polyethylenimine (PEI). In addition, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection.nnnSTATEMENT OF SIGNIFICANCEnIn this study, we developed functional quantum dot (QD) nanoplexes by sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation of PEG-coated CdSe/ZnS QDs as the gene carrier of siRNA to study the effect of SOX9 RNA interference on the chondrogenic differentiation of MSCs. This study confirmed the importance of SOX9 in chondrogenesis, as evidenced by the findings that SOX9 knockdown significantly inhibited the expression of cartilage-specific markers including acan and col2a1 in MSCs and further delayed cartilage repair. Moreover, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection.

Material‑induced chondrogenic differentiation of mesenchymal stem cells is material‑dependent

Certain materials may mimic natural cartilage to provide an amenable cellular microenvironment for the chondrogenic differentiation of mesenchymal stem cells. The chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has been demonstrated to be induced by collagen-based hydrogels in vivo, but whether the induction is material-driven or self-differentiation has not been elucidated. In the present study, BMSCs were encapsulated in porous materials, namely, a biphasic calcium phosphate ceramic (BCP), silk fibroin protein matrix (SFP) and collagen sponge (CS), to further study the chondrogenic effects of various materials. Diffusion chambers that allow the body fluid to permeate and deter the host cells from invasion were also loaded with the cell-scaffold constructs. Chambers containing the scaffold-BMSC composites were implanted subcutaneously in the dorsa of rabbits. The specimens in the chamber were harvested for histological and immunohistochemical analyses eight weeks after implantation. The results showed that no chondrogenic differentiation of the BMSCs occurred when the BMSCs were encapsulated in BCP, SFP and CS, indicating that chondrogenesis induced by materials is material-dependent and that these particular porous materials are not suitable for inducing chondrogenesis. However, the diffusion chamber was effective in preventing host immune rejection, host cell invasion and vascular invasion. The results are likely to serve as a valuable clinical reference when selecting an appropriate scaffold for cartilage repair.

Comparative study of collagen hydrogels modified in two ways using the model of ectopic cartilage construction with diffusion-chamber in immunocompetent host

Purpose For scaffolds in cartilage tissue engineering, it is the principle to design the materials with both favorable mechanical and biological property. Methods In this article, collagen hydrogels modified by two ways to improve mechanical strength were applied for in vivo cartilage reconstruction: one is collagen-alginate hydrogel (CAH) representative of mixture, the other is collagen hydrogel crosslinked by genipin (CGH). To investigate the biological activities of the two materials, it was designed as: scaffolds loaded with allogenous chondrocytes were encased in diffusion chamber, and then implanted subcutaneously in SD rats for 8 weeks. Results Histologic, immunohistochemical, and RT-PCR results showed that collagen type II and GAG, indicator of cartilage extracellular matrix (ECM) was highly expressed in constructs of chondrocyte-CAH. Significantly lower cell density and expression of cartilage specific protein were shown in constructs of chondrocyte-CGH than that in chondrocyte-CAH. This demonstrated that CAH may provide a more favorable environment for cartilage reconstruction. In addition, the model with diffusion chamber technique was viable for evaluation of scaffolds for in vivo cartilage engineering in immunocompetent host. Instead, directly reconstruction of ectopic cartilage without diffusion chamber suffered from damaged tissue and less neo-cartilage matrix formed. Conclusions In conclusion, CAH is realistic as scaffold for in vivo cartilage tissue engineering with both satisfactory mechanical properties and biomimetic activity. Also, the model with diffusion chamber to reconstruct ectopic cartilage in immunocompetent animals is promising for evaluation of scaffolds. This study provided a new insight for in vivo cartilage tissue engineering.

The role of Sox9 in collagen hydrogel-mediated chondrogenic differentiation of adult mesenchymal stem cells (MSCs)

Sox9 is a transcription factor that regulates chondrogenesis, but its role in the chondrogenic differentiation of mesenchymal stem cells (MSCs) triggered by materials is poorly understood. In this study, we investigated the effect of Sox9 interference on collagen-induced chondrogenesis and further collagen-based therapies for cartilage defects. In this paper, MSCs were infected with a vector carrying the Sox9 promoter and related markers were detected. A lentivirus-mediated vector targeting the silencing of the Sox9 gene was used in bone marrow-derived MSCs prior to being encapsulated in a collagen hydrogel. The collagen hydrogel as a sole inducer was also compared with transforming growth factor-β1 (TGF-β1). Before being implanted into the articular cartilage defect in rats, the cell-hydrogel pellets were cultured in vitro for 14 days. The effect of Sox9 transfection on cell proliferation was evaluated by measuring the total DNA content. Safranin-O staining and a biochemistry assay were performed to assess the synthesis and secretion of glycosaminoglycan (GAG) of MSCs. The real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was performed to detect the gene expression levels of Col1a1, Col2a1, Acan and Sox9. The protein expression of collagen type II and collagen type I was analyzed by immunohistochemical analysis. Collagen alone significantly increased the luciferase activity of the Sox9 promoter, which was in parallel with the upregulation of cartilage specific markers. In vitro, the chondrogenic differentiation ability of MSCs was greatly inhibited after Sox9 interference, both in the collagen and TGF-β1-induced groups. In vivo, a further study showed that cartilage regeneration was arrested by using transfected MSCs with an injectable collagen gel or induced by TGF-β1. The results indicated that collagen may mediate Sox9 expression by providing a biomimetic microenvironment favoring cell condensation prior to chondrogenesis. The role of Sox9 regulation by materials is similar to that by growth factors, suggesting that well-designed scaffolds may replace growth factors in chondrogenesis. Thus, interventions targeting Sox9 may help improve articular cartilage repair.