Long Bi

Effects of different cross-linking conditions on the properties of genipin-cross-linked chitosan/collagen scaffolds for cartilage tissue engineering

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended.

Gelatin Microspheres Containing TGF-β3 Enhance the Chondrogenesis of Mesenchymal Stem Cells in Modified Pellet Culture

The study is to investigate the chondrogenesis of a kind of modified cell pellet formed using mesenchymal stem cells (MSCs) and gelatin microspheres containing transforming growth factor beta3 (TGF-beta3). The gelatin microspheres loaded with TGF-beta3 (MS-TGF) were prepared and showed the controlled release of cytokine in a biphasic fashion. Then the mixture of MSCs and MS-TGF was centrifuged to form pellet. The pellet was cultured over 4 weeks to determine the effects of MS-TGF on cartilage matrix production by biochemical analysis, immunohistochemistry staining, and Western blot test. The transcription level of cartilage-related genes was also evaluated by real-time quantitative RT-PCR assay. After 4 weeks of culture, the MSCs were distributed uniformly in the pellet and had good viability. Cells showed faster proliferation and higher DNA content compared to MSCs in a conventional pellet. The production of collagen and glycosaminoglycan also increased significantly. The immunohistochemistry staining and alcian blue staining confirmed the synthesis of cartilage extracellular matrix (ECM). Furthermore, the differentiated MSCs located in lacunae within the metachromatic staining matrix exhibited the typical chondrocyte morphology. The chondrogenic differentiation of MSCs was proved by the expression of collagen II gene in mRNA and protein level. The results indicate that MS-TGF can induce chondrogenic differentiation of MSCs and increase cartilage ECM production, which result in a bigger cartilage pellet. In conclusion, this modified pellet culture can provide an easy and effective way to construct the tissue-engineered cartilage in vitro.

Enhancing the bioactivity of Poly(lactic-co-glycolic acid) scaffold with a nano-hydroxyapatite coating for the treatment of segmental bone defect in a rabbit model

Purpose Poly(lactic-co-glycolic acid) (PLGA) is excellent as a scaffolding matrix due to feasibility of processing and tunable biodegradability, yet the virgin scaffolds lack osteoconduction and osteoinduction. In this study, nano-hydroxyapatite (nHA) was coated on the interior surfaces of PLGA scaffolds in order to facilitate in vivo bone defect restoration using biomimetic ceramics while keeping the polyester skeleton of the scaffolds. Methods PLGA porous scaffolds were prepared and surface modification was carried out by incubation in modified simulated body fluids. The nHA coated PLGA scaffolds were compared to the virgin PLGA scaffolds both in vitro and in vivo. Viability and proliferation rate of bone marrow stromal cells of rabbits were examined. The constructs of scaffolds and autogenous bone marrow stromal cells were implanted into the segmental bone defect in the rabbit model, and the bone regeneration effects were observed. Results In contrast to the relative smooth pore surface of the virgin PLGA scaffold, a biomimetic hierarchical nanostructure was found on the surface of the interior pores of the nHA coated PLGA scaffolds by scanning electron microscopy. Both the viability and proliferation rate of the cells seeded in nHA coated PLGA scaffolds were higher than those in PLGA scaffolds. For bone defect repairing, the radius defects had, after 12 weeks implantation of nHA coated PLGA scaffolds, completely recuperated with significantly better bone formation than in the group of virgin PLGA scaffolds, as shown by X-ray, Micro-computerized tomography and histological examinations. Conclusion nHA coating on the interior pore surfaces can significantly improve the bioactivity of PLGA porous scaffolds.

The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells.

Icariin had been reported as a potential agent for osteogenesis, but the dose-effect relationship needed further research to realize the clinical application of icariin. We isolated and purified human bone mesenchymal stem cells (hBMSCs) and stimulated them with different concentrations of icariin. The cytotoxicity of icariin was evaluated by the methylthiazolytetrazolium (MTT) assay method. The proliferation and osteogenic differentiation of such hBMSCs were investigated for different concentrations of icariin. We found that icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of hBMSCs in a suitable concentration range from 10−9 M to 10−6 M, but at concentrations above 10−5 M, the cytotoxicity limited its use. The extremely low cost of icariin and its high abundance make it appealing for bone regeneration.

A combined chitosan/nano-size hydroxyapatite system for the controlled release of icariin

Icariin, a plant-derived flavonol glycoside, has been proved as an osteoinductive agent for bone regeneration. For this reason, we developed an icariin-loaded chitosan/nano-sized hydroxyapatite (IC–CS/HA) system which controls the release kinetics of icariin to enhance bone repairing. First, by Fourier transform infrared spectroscopy, we found that icariin was stable in the system developed without undergoing any chemical changes. On the other hand, X-ray diffraction, scanning electron microscopy and mechanical test revealed that the introduction of icariin did not remarkably change the phase, morphology, porosity and mechanical strength of the CS/HA composite. Then the hydrolytic degradation and drug release kinetics in vitro were investigated by incubation in phosphate buffered saline solution. The results indicated that the icariin was released in a temporally controlled manner and the release kinetics could be governed by degradation of both chitosan and hydroxyapatite matrix. Finally the in vitro bioactivity assay revealed that the loaded icariin was biologically active as evidenced by stimulation of bone marrow derived stroma cell alkaline phosphatase activity and formation of mineralized nodules. This successful IC–CS/HA system offers a new delivery method of osteoinductive agents and a useful scaffold design for bone regeneration.

Low-Temperature Additive Manufacturing of Biomimic Three-Dimensional Hydroxyapatite/Collagen Scaffolds for Bone Regeneration.

Low-temperature additive manufacturing (AM) holds promise for fabrication of three-dimensional (3D) scaffolds containing bioactive molecules and/or drugs. Due to the strict technical limitations of current approaches, few materials are suitable for printing at low temperature. Here, a low-temperature robocasting method was employed to print biomimic 3D scaffolds for bone regeneration using a routine collagen-hydroxyapatite (CHA) composite material, which is too viscous to be printed via normal 3D printing methods at low temperature. The CHA scaffolds had excellent 3D structure and maintained most raw material properties after printing. Compared to nonprinted scaffolds, printed scaffolds promoted bone marrow stromal cell proliferation and improved osteogenic outcome in vitro. In a rabbit femoral condyle defect model, the interconnecting pores within the printed scaffolds facilitated cell penetration and mineralization before the scaffolds degraded and enhanced repair, compared to nonprinted CHA scaffolds. Additionally, the optimal printing parameters for 3D CHA scaffolds were investigated; 600-μm-diameter rods were optimal in terms of moderate mechanical strength and better repair outcome in vivo. This low-temperature robocasting method could enable a variety of bioactive molecules to be incorporated into printed CHA materials and provides a method of bioprinting biomaterials without compromising their natural properties.

FFAs-ROS-ERK/P38 pathway plays a key role in adipocyte lipotoxicity on osteoblasts in co-culture

The accumulation of adipocytes in bone marrow is common in a variety of pathophysiological conditions, including obesity, insulin resistance, type 2 diabetes, and aging. Adipocytes in bone marrow exhibit severe adverse effect on osteoblast differentiation, proliferation, and function. However, the molecular mechanism of adipocytes lipotoxicity on osteoblasts is still far from completely understood. The present study was designed to investigate the signaling pathway responsible for adipocytes lipotoxicity on osteoblasts. Using a co-culture system, we have identified that free fatty acids (FFAs) released by the adipocytes inhibited osteoblasts proliferation and function and induced osteoblasts apoptosis, evidenced by decreased cell viability/proliferation, ALP activity, expression of runt-related transcription factor 2 (RunX2), type I collagen (ColA1) and osteocalcin and alizarin red staining. Dexamethasone (Dex) promoted the inhibitory effect of adipocytes on osteoblasts through stimulating FFAs release. Dex-exacerbated FFAs release from adipocytes contributes to reactive oxygen species (ROS) production. In the co-culture system, the phosphorylation of extracellular signal-regulated kinase (ERK)/P38 was increased and inhibition of ERK/P38 significantly suppressed adipocytes lipotoxicity. FFAs-generated ROS was responsible for adipocytes-induced activation of ERK/P38 signaling. In conclusion, FFAs-ROS-ERK/P38 pathway plays a key role in adipocyte lipotoxicity on osteoblasts in co-culture. The evidence provides new insights into the mechanisms underlying the lipotoxic effect of adipocytes on bone within the marrow microenvironment and prevention of lipotoxicity on bone metabolism.

Neuropeptide Substance P Improves Osteoblastic and Angiogenic Differentiation Capacity of Bone Marrow Stem Cells In Vitro

Our previous work showed that implanting a sensory nerve or vascular bundle when constructing vascularized and neurotized bone could promote bone osteogenesis in tissue engineering. This phenomenon could be explained by the regulatory function of neuropeptides. Neuropeptide substance P (SP) has been demonstrated to contribute to bone growth by stimulating the proliferation and differentiation of bone marrow stem cells (BMSCs). However, there have been no prior studies on the association between Wnt signaling and the mechanism of SP in the context of BMSC differentiation. Our results have shown that SP could enhance the differentiation of BMSCs by activating gene and protein expression via the Wnt pathway and by translocating β-catenin, which can be inhibited by Wnt signaling blocker treatment or by the NK-1 antagonist. SP could also increase the growth factor level of bone morphogenetic protein-2 (BMP-2). Additionally, SP could enhance the migration ability of BMSCs, and the promotion of vascular endothelial growth factor (VEGF) expression by SP has been studied. In conclusion, SP could induce osteoblastic differentiation via the Wnt pathway and promote the angiogenic ability of BMSCs. These results indicate that a vascularized and neurotized tissue-engineered construct could be feasible for use in bone tissue engineering strategies.

The potential use of allogeneic platelet-rich plasma for large bone defect treatment: immunogenicity and defect healing efficacy.

Autologous platelet-rich plasma (PRP) has been extensively investigated for large bone defect treatment, but its clinical application is harassed by controversial outcome, due to highly variable PRP quality among patients. Alternatively, allogeneic PRP from well-characterized donors cannot only generate more consistent and reliable therapeutic effect but also avoid harvesting large quantities of blood, an additional health burdens to patients. However, the use of allogeneic PRP for bone defect treatment is generally less investigated, especially for its immunogenicity in such application. Here, we meticulously investigated the immunogenicity of allogeneic PRP and evaluated its healing efficacy for critical-sized defect treatment. Allogeneic PRP contained 4.1-fold and 2.7- to 4.9-fold higher amount of platelets and growth factors than whole blood, respectively. The intramuscular injection of allogeneic PRP to rabbits did not trigger severe and chronic immunoresponse, evidenced by little change in muscular tissue microstructure and CD4+/CD8+ T lymphocyte subpopulation in peripheral blood. The implantation of allogeneic PRP/deproteinized bone matrix (DPB) constructs (PRP + DPB) successfully bridged 1.5-cm segmental radial defects in rabbits, achieving similar healing capacity as autologous MSC/DPB constructs (MSC + DPB), with greater bone formation (1.1–1.5x, p < 0.05) and vascularization (1.3–1.6x, p < 0.05) than DPB alone, shown by histomorphometric analysis, bone mineral density measurement, and radionuclide bone imaging. Furthermore, the implantation of both allogeneic PRP- and autologous MSC-mediated DPB constructs (PRP + MSC + DPB) resulted in the most robust bone regeneration (1.2–2.1x, p < 0.05) and vascularization (1.3–2.0x, p < 0.05) than others (PRP + DPB, MSC + DPB, or DPB alone). This study has demonstrated the promising use of allogeneic PRP for bone defect treatment with negligible immunogenicity, great healing efficacy, potentially more consistent quality, and no additional health burden to patients; additionally, the synergetic enhancing effect found between allogeneic PRP and autologous MSCs may shed a light on developing new therapeutic strategies for large bone defect treatment.

Increased levels of calcitonin gene-related peptide in serum accelerate fracture healing following traumatic brain injury

The mechanisms responsible for the phenomenon of an accelerated speed of fracture healing in patients with traumatic brain injury (TBI) remain unclear. The present study was performed to test the hypothesis that TBI causes changes in calcitonin gene-related peptide (CGRP) levels in sera that enhance fracture healing. A standard closed femoral fracture was produced in rats, which were subjected to additional closed head trauma. The fracture healing was assessed 4 and 8 weeks later using micro-CT. Sera, brain tissues and muscles surrounding the fracture sites collected at 24, 48, 72 and 168 h after injury were used to detect the expression of CGRP using ELISA, immunohistochemistry and RT-PCR. Micro-CT demonstrated that fracture healing and mineralization in the TBI-fracture group occurred earlier compared to the fracture-only group. ELISA analysis revealed a high concentration of CGRP in the TBI-fracture group (P<0.05), and immunohistochemistry assay and RT-PCR analysis revealed a significant increase in CGRP in the brain and muscle of the TBI-fracture group at 168 h after fracture (P<0.001). Our results indicate that the mechanism for the enhancement of fracture-healing secondary to traumatic brain injury is correlated to the high levels of CGRP, which may be released from the brain tissue into the serum.