Xuening Chen

Fabrication and cellular biocompatibility of porous carbonated biphasic calcium phosphate ceramics with a nanostructure.

Microwave heating was applied to fabricate interconnective porous structured bodies by foaming as-synthesized calcium-deficient hydroxyapatite (Ca-deficient HA) precipitate containing H(2)O(2). The porous bodies were sintered by a microwave process with activated carbon as the embedding material to prepare nano- and submicron-structured ceramics. By comparison, conventional sintering was used to produce microstructured ceramics. The precursor particles and bulk ceramics were characterized by transmission electron microscopy (TEM), dynamic light scattering, scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier-transformed infrared spectroscopy (FTIR) and mechanical testing. TEM micrographs and assessment of the size distribution showed that the needle-like precursor particles are on the nanoscale. SEM observation indicated that the ceramics formed by microwave sintering presented a structure of interconnective pores, with average grain sizes of approximately 86 and approximately 167nm. XRD patterns and FTIR spectra confirmed the presence of carbonated biphasic calcium phosphate (BCP), and the mechanical tests showed that the ceramics formed by microwave sintering had a compressive strength comparable to that obtained by conventional methods. Rat osteoblasts were cultured on the three kinds of BCP ceramics to evaluate their biocompatibility. Compared with the microscale group formed by conventional sintering, MTT assay and ALP assay showed that nanophase scaffolds promoted cell proliferation and differentiation respectively, and SEM observation showed that the nanoscale group clearly promoted cell adhesion. The results from this study suggest that porous carbonated biphasic calcium phosphate ceramics with a nanostructure promote osteoblast adhesion, proliferation and differentiation. In conclusion, porous carbonated BCP ceramics with a nanostructure are simple and quick to prepare using microwaves and compared with those produced by conventional sintering, may be better bone graft materials.

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.

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.

Role of biphasic calcium phosphate ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs

The inflammatory reaction initiates fracture healing and could play a role in the osteoinductive effect of calcium phosphate (CaP) ceramics, which has been widely confirmed; however, the underlying mechanism has not been fully elucidated. In this study, various signaling molecules from macrophages under the stimulation of osteoinductive biphasic calcium phosphate (BCP) ceramic and its degradation products were examined and evaluated for their influence on the migration and osteoblastic differentiation of mesenchymal stem cells (MSCs). The results of cellular experiments confirmed that the gene expression of most inflammatory factors (IL-1, IL-6 and MCP-1) and growth factors (VEGF, PDGF and EGF) by macrophages were up-regulated to varying degrees by BCP ceramic and its degradation products. Cell migration tests demonstrated that the conditioned media (CMs), which contained abundant signaling molecules secreted by macrophages cultured on BCP ceramic and its degradation products, promoted the migration of MSCs. qRT-PCR analysis indicated that CMs promoted the gene expression of osteogenic markers (ALP, COL-I, OSX, BSP and OPN) in MSCs. ALP activity and mineralization staining further confirmed that CMs promoted the osteoblastic differentiation of MSCs. The present study confirmed the correlation between the inflammatory reaction and osteoinductive capacity of BCP ceramic. The ceramic itself and its degradation products can induce macrophages to express and secrete various signaling molecules, which then recruit and promote the MSCs to differentiate into osteoblasts. Compared with BCP conditioned media, degradation particles played a more substantial role in this process. Thus, inflammation initiated by BCP ceramic and its degradation products could be necessary for osteoinduction by the ceramic. STATEMENT OF SIGNIFICANCE It is known that the inflammatory reaction initiates fracture healing. The aim of this study was to examine whether osteoinductive BCP ceramics could cause macrophages to change their secretion patterns and whether the secreted cytokines could affect migration and osteoblastic differentiation of MSCs. Moreover, the duration of inflammation could be influenced by the local ionic environment and the degradation products of the implant. Our experimental results revealed the correlation between the inflammatory reaction and osteoinductive capacity of BCP ceramic. The ceramic itself and its degradation products can induce macrophages to express and secrete various signaling molecules, which then recruit and promote the MSCs to differentiate into osteoblasts. Compared with ionic microenvironment, degradation particles played a more substantial role in this process. Therefore, the appropriate inflammation initiated by BCP ceramic and its degradation products could be essential for osteoinduction by the ceramic. We believe that the present study improves the understanding of the effect of biomaterial-mediated inflammation on MSC migration and differentiation and established a preliminary correlation between the immune system and osteoinduction by biomaterials.

Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice

Phase composition played a key role in the biodegradation of calcium phosphate ceramics (CaP), which in turn influences the osteoinductive ability. The in vivo biological mechanism is still poorly understood. In this study, four types of porous CaP ceramics were investigated, namely, hydroxyapatite (HA), β-tricalcium phosphate (TCP), and biphasic calcium phosphates BCP1 and BCP2, with HA to β-TCP ratios of 70/30 and 30/70, respectively. The four types of ceramics were implanted into thigh muscle of mice for 16weeks. Longitudinal ectopic bone formation process at gene, protein, and tissue level induced by the material was assessed. Histological analysis revealed that BCP2 was the only group that had promoted new bone formation after 16weeks. In micro-CT analysis of biodegradation, the BCP2 group had the least increment of porosity due to the new bone formation, resulting in a significant elevation in material density. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression patterns. The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Among the target genes, Osterix and type I collagen were activated successively. The osteoinductive BCP2 group showed earlier and significantly higher peaks in BMP2, BMPR1A, and OPG expressions than non-bone forming groups. These findings revealed that the occurrence time and magnitude of these osteogenetic gene expression peaks can be crucial in the osteoinduction process.

Selective effect of hydroxyapatite nanoparticles on osteoporotic and healthy bone formation correlates with intracellular calcium homeostasis regulation

Adequate bone substitutes osseointegration has been difficult to achieve in osteoporosis. Hydroxyapatite of the osteoporotic bone, secreted by pathologic osteoblasts, had a smaller crystal size and lower crystallinity than that of the normal. To date, little is known regarding the interaction of synthetic hydroxyapatite nanoparticles (HANPs) with osteoblasts born in bone rarefaction. The present study investigated the biological effects of HANPs on osteoblastic cells derived from osteoporotic rat bone (OVX-OB), in comparison with the healthy ones (SHM-OB). A selective effect of different concentrations of HANPs on the two cell lines was observed that the osteoporotic osteoblasts had a higher tolerance. Reductions in cell proliferation, ALP activity, collagen secretion and osteoblastic gene expressions were found in the SHM-OB when administered with HANPs concentration higher than 25µg/ml. In contrast, those of the OVX-OB suffered no depression but benefited from 25 to 250µg/ml HANPs in a dose-dependent manner. We demonstrated that the different effects of HANPs on osteoblasts were associated with the intracellular calcium influx into the endoplasmic reticulum. The in vivo bone defect model further confirmed that, with a critical HANPs concentration administration, the osteoporotic rats had more and mechanically matured new bone formation than the non-treated ones, whilst the sham rats healed no better than the natural healing control. Collectively, the observed epigenetic regulation of osteoblastic cell function by HANPs has significant implication on defining design parameters for a potential therapeutic use of nanomaterials. STATEMENT OF SIGNIFICANCE In this study, we investigated the biological effects of hydroxyapatite nanoparticles (HANPs) on osteoporotic rat bone and the derived osteoblast. Our findings revealed a previously unrecognized phenomenon that the osteoporotic individuals could benefit from higher concentrations of HANPs, as compared with the healthy individuals. The in vivo bone defect model confirmed that, with a critical HANPs concentration administration, the osteoporotic rats had more mechanically matured new bone formation than the non-treated ones, whilst the sham rats healed no better than the natural healing control. The selective effect of HANPs might be associated with the intracellular calcium influx into the endoplasmic reticulum. Collectively, the observed epigenetic regulation by HANPs has significant implication on defining design parameters for a potential therapeutic use of nanomaterials in a pathological condition.

Administration duration influences the effects of low-magnitude, high-frequency vibration on ovariectomized rat bone

Low‐magnitude, high‐frequency vibration (LMHFV) has been proposed as a non‐drug anti‐osteoporosis treatment. However, the influence of administration duration on its effect is seldom investigated. In this study, the effect of 16‐week LMHFV (0.3 g, 30 Hz, 20 min/day) on the bone mineral densities (BMDs), bone mechanical properties, and cellular responses of osteoporotic and healthy rats was examined by in vivo peripheral quantitative computed tomography (pQCT), fracture tests, cell assays, and mRNA quantification. Forty‐eight adult rats were equally assigned to sham surgery (SHM), sham surgery with LMHFV (SHM+V), ovariectomy (OVX), and ovariectomy with LMHFV (OVX+V) groups. At week 8, LMHFV ameliorated ovariectomy‐induced deterioration of trabecular bone, with a significantly higher tibia trabecular BMD (+11.2%) being noted in OVX+V rats (vs. OVX). However, this positive effect was not observed at later time points. Furthermore, 16 weeks of LMHFV caused significant reductions in the vertebral mean BMD (−13.0%), trabecular BMD (−15.7%), and maximum load (−21.5%) in OVX+V rats (vs. OVX). Osteoblasts derived from osteoporotic rat bone explants showed elevated BSP and OSX mRNA expression induced by LMHFV on day 1. However, no further positive effect on osteoblastic mRNA expression, alkaline phosphatase activity, or calcium deposition was observed with prolonged culture time. A higher ratio of RANKL/OPG induced by LMHFV suggests that osteoclastogenesis may be activated. Together, these results demonstrate that administration duration played an important role in the effect of LMHFV. Early exposure to LMHFV can positively modulate osteoporotic bone and osteoblasts; however, the beneficial effect seems not to persist over time.

Temperature and ion dual responsive biphenyl-dipeptide supramolecular hydrogels as extracellular matrix mimic-scaffolds for cell culture applications

Stimuli-responsive supramolecular hydrogels composed of aromatic short peptide gelators have attracted intensive attention in the field of biomedicine because of their stable chemical structure, simple and convenient synthetic route and intelligent response to external stimuli. In this paper, several dipeptides were coupled to biphenylacetic acid (BPAA) to generate aromatic short peptide compounds through the standard solid phase peptide synthesis. These BPAA-dipeptide compounds presented clearly different gelation behaviors from the generally employed Fmoc-dipeptide and Nap-dipeptide compounds, but only BPAA-diphenylalanine was able to form homogeneous and transparent hydrogels through temperature switching or ion induction. Utilizing the biphenyl group not only expanded the scope of aromatic molecules serving as building blocks of aromatic short peptide gelators but also demonstrated the critical role of aromatic molecules in the self-assembling process. Moreover, supramolecular hydrogels initiated by heating-cooling or salt addition could be exploited as extracellular matrix (ECM) mimic scaffolds to support the adhesive growth and proliferation of L929 cells in 2D/3D culture under physiological conditions, demonstrating their potential applications in regenerative medicine.

Stabilization of Ca-deficient hydroxyapatite in biphasic calcium phosphate ceramics by adding alginate to enhance their biological performances

It is of significance to further improve the bioactivity of existing calcium phosphate (Ca-P) biomaterials to satisfy the needs of regenerative medicine. Due to its compositional similarity to natural bone mineral, calcium-deficient hydroxyapatite (CDHA) is supposed to possess excellent bioactivity. However, it is difficult to fabricate Ca-P ceramics with a high amount of CDHA, as CDHA is easy to decompose during the sintering process. The present study introduced an effective approach to stabilize CDHA in biphasic calcium phosphate (BCP) ceramics by adding alginate, and investigated the roles of CDHA in their biological performances. The characterization of the phase composition, crystal structure, and functional group demonstrated that the addition of alginate could obviously inhibit CDHA decomposition to attain novel BCP ceramics with a high CDHA content (BCP-A), which could better mimic the inorganic composition of natural bones as compared with the conventional BCP ones (BCP-C). In vitro studies suggested BCP-A showed better bioactivity and osteoinductive capacity than BCP-C, as evidenced by the increased serum protein adsorption, better bone-like apatite formation and cell spreading, and promoted osteogenic differentiation. In vivo intramuscular implantation further confirmed that BCP-A could induce more ectopic bone formation than BCP-C, suggesting BCP-A had a stronger osteoinductivity. Altogether, this study demonstrates that the stabilization of CDHA in BCP ceramics by adding alginate offers a promising principle for designing regenerative biomaterials to process superior biological performances.