The objective of the present study was to evaluate the capacity of a tissue-engineered bone complex of recombinant human bone morphogenetic protein 2 (rhBMP-2)-mediated dental pulp stem cells (DPSCs) and nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA) to reconstruct critical-size alveolar bone defects in New Zealand rabbit. Autologous DPSCs were isolated from rabbit dental pulp tissue and expanded ex vivo to enrich DPSCs numbers, and then their attachment and differentiation capability were evaluated when cultured on the culture plate or nHAC/PLA. The alveolar bone defects were treated with nHAC/PLA, nHAC/PLA+rhBMP-2, nHAC/PLA+DPSCs, nHAC/PLA+DPSCs+rhBMP-2, and autogenous bone (AB) obtained from iliac bone or were left untreated as a control. X-ray and a polychrome sequential fluorescent labeling were performed postoperatively and the animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. Our results showed that DPSCs expressed STRO-1 and vementin, and favored osteogenesis and adipogenesis in conditioned media. DPSCs attached and spread well, and retained their osteogenic phenotypes on nHAC/PLA. The rhBMP-2 could significantly increase protein content, alkaline phosphatase activity/protein, osteocalcin content, and mineral formation of DPSCs cultured on nHAC/PLA. The X-ray graph, the fluorescent, histological observation, and histomorphometric analysis showed that the nHAC/PLA+DPSCs+rhBMP-2 tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than nHAC/PLA, nHAC/PLA+rhBMP-2, and nHAC/PLA+DPSCs, or even autologous bone. Implanted DPSCs' contribution to new bone was detected through transfected eGFP genes. Our findings indicated that stem cells existed in adult rabbit dental pulp tissue. The rhBMP-2 promoted osteogenic capability of DPSCs as a potential cell source for periodontal bone regeneration. The nHAC/PLA could serve as a good scaffold for autologous DPSC seeding, proliferation, and differentiation. The tissue-engineered bone complex with nHAC/PLA, rhBMP-2, and autologous DPSCs might be a better alternative to autologous bone for the clinical reconstruction of periodontal bone defects.

Background-Embryonic stem (ES) cells can terminally differentiate into all types of somatic cells and are considered a promising source of seed cells for tissue engineering. However, despite recent progress in in vitro differentiation and in vivo transplantation methodologies of ES cells, to date, no one has succeeded in using ES cells in tissue engineering for generation of somatic tissues in vitro for potential transplantation therapy. Methods and Results-ES-D3 cells were cultured in a slow-turning lateral vessel for mass production of embryoid bodies.The embryoid bodies were then induced to differentiate into cardiomyocytes in a medium supplemented with 1% ascorbic acid. The ES cell-derived cardiomyocytes were then enriched by Percoll gradient centrifugation.

Periodontal bone defects occur in a wide variety of clinical situations. Adult stem cell- and biomaterial-based bone tissue regeneration are a promising alternative to natural bone grafts. Recent evidence has demonstrated that two populations of adult bone marrow mesenchymal stromal cells (BMSCs) can be distinguished based on their embryonic origins. These BMSCs are not interchangeable, as bones preferentially heal using cells that share the same embryonic origin. However, the feasibility of tissue engineering using human craniofacial BMSCs was unclear. The goal of this study was to explore human craniofacial BMSC-based therapy for the treatment of localized mandibular defects using a standardized, minimally invasive procedure. The BMSCs' identity was confirmed. Scanning electron microscopy, a cell proliferation assay, and supernatant detection indicated that the nHAC/PLA provided a suitable environment for aBMSCs. Real-time PCR and electrochemiluminescence immunoassays demonstrated that osteogenic markers were upregulated by osteogenic preinduction. Moreover, in a rabbit critical-size mandibular bone defect model, total bone formation in the nHAC/PLA + aBMSCs group was significantly higher than in the nHAC/PLA group but significantly lower than in the nHAC/PLA + preinduced aBMSCs. These findings demonstrate that this engineered bone is a valid alternative for the correction of mandibular bone defects.

Insulin is considered to be a classical central regulator of energy homeostasis. Recently, the effect of insulin on bone has gained a lot of attention, but little attention has been paid to the application in bone tissue engineering. In this study, porous nanohydroxyapatite/collagen (nHAC) scaffolds incorporating poly lactic-co-glycolic acid (PLGA) particles were successfully developed as an insulin delivery platform for bone regeneration. Bioactive insulin was successfully released from the PLGA particles within the scaffold, and the size of the particles as well as the release kinetics of the insulin could be efficiently controlled through Shirasu porous glass premix membrane emulsification technology. It was indicated that the nHAC/PLGA composite scaffolds possessed favorable mechanical and structural properties for cell adhesion and proliferation, as well as the differentiation into osteoblasts. It was also demonstrated that the nHAC/PLGA scaffolds implanted into a rabbit critical-size mandible defect possessed tissue compatibility and higher bone restoration capacity compared with the defects that were filled with or without nHAC scaffolds. Furthermore, the in vivo results showed that the nHAC/PLGA scaffolds which incorporated insulin-loaded microspheres with a size of 1.61 μm significantly accelerated bone healing compared with two other composite scaffolds. Our study indicated that the local insulin released at the optimal time could substantially and reproducibly improve bone repair.

This study investigated the effects of a newly-developed scaffold, nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA), on the attachment, proliferation and osteogenic capability of dog periodontal ligament stem cells (PDLSCs) in vitro and in vivo. Hydroxyapatite/tricalcium phosphate (HA/TCP), a commonly used bone substitute, was used as a positive control. PDLSCs isolated from dog molar were incubated in an osteogenic medium to evaluate their osteogenic differentiation in vitro, and then seeded onto nHAC/PLA and HA/TCP scaffolds. In vitro cell attachment, proliferation and differentiation were assessed by scanning electron microscopy (SEM), cell counting, 3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium and alkaline phosphate activity, and reverse transcription-polymerase chain reaction, respectively. Finally, the constructs were implanted subcutaneously into dogs to investigate their osteogenic capacity. After osteogenic induction for 21 days, PDLSCs differentiated into osteogenic lineage, as indicated by the expressions of osteoblastic differentiation genes CoL-I, OCN and OPN mRNA, and the formation of mineral deposits. When seeded onto scaffolds, the cells attached and spread well, and retained their osteogenic phenotypes on both scaffolds. Comparatively, cell number and proliferative viability on nHAC/PLA constructs were greater than those on HA/TCP constructs (P < 0.05). Histological results showed that new bone and osteoid was formed in both groups, and histomorphometric analysis demonstrated that the amount of newly formed bone in the nHAC/PLA group was higher than that in the HA/TCP group (P < 0.05). This study suggests that nHAC/PLA can be used as a potent scaffold for alveolar bone regeneration.