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The scaffold microstructure has a great impact on cell functions in tissue engineering. Herein, the PLLA scaffolds with hierarchical fiber size and pore size were successfully fabricated by thermal-induced phase separation or combined thermal-induced phase separation and salt leaching methods. The PLLA scaffolds were fabricated as microfibrous scaffolds, microfibrous scaffolds with macropores (50–350 µm), nanofibrous scaffolds with micropores (100 nm to 10 µm), and nanofibrous scaffolds with both macropores and micropores by tailoring selective solvents for forming different fiber size and pre-sieved salts for creating controlled pore size. Among the four kinds of PLLA scaffolds, the nanofibrous scaffolds with both macropores and micropores provided a favorable microenvironment for protein adsorption, cell proliferation, and cell infiltration. The results further confirmed the significance of fiber size and pore size on the biological properties, and a scaffold with both micropores and macropores, and a nanofibrous matrix might have promising applications in bone tissue engineering.

Human adipose-derived stem cells (ASCs) have the ability to differentiate into osteoblasts and thus the potential therapeutic use to tissue-engineer bone, so a reliable method for cell storage is necessary. The aim of this study was to determine whether a simple method of cryopreservation with 10% Me(2)SO as a protectant had an effect on proliferation potential and osteogenic differentiation of ASCs isolated from fresh human adipose tissue. ASCs were harvested from 6 human lipoaspirates and each was halved for either cryopreservation in liquid nitrogen for 2 weeks or for control culture. Cells from the second-passage were plated at a density of 5000cells/well in 24-well plates and cultured with or without osteogenic media for 14 days. Cell surface antigens were used to identify the cryopreserved ASCs by flow cytometry. The proliferation rate of both populations was evaluated using a cell DNA assay. To detect osteogenic differentiation of both the cryopreserved and non-cryopreserved populations, determination of osteoblastic protein production (alkaline phosphatase and osteocalcin) and excellular matrix calcification (calcium content) was applied. The expression of osteoblastic-associated genes was also analyzed using reverse-transcription polymerase chain reaction. These results demonstrate that cryopreservation has no effect on the phenotype, proliferation or osteogenic differentiation of human ASCs, showing cryopreserved human ASCs might be applied for bone tissue engineering.

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel beta-tricalcium phosphate (beta-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous beta-TCP scaffolds with pores of 300-500 microm in size were prepared by the polymeric sponge method. beta-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p0.05). These results indicated that the novel porous beta-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In beta-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in beta-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous beta-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

Item does not contain fulltext The reconstruction of large craniofacial defects remains a significant clinical challenge. The complex geometry of facial bone and the lack of suitable donor tissue often hinders successful repair. One strategy to address both of these difficulties is the development of an in vivo bioreactor, where a tissue flap of suitable geometry can be orthotopically grown within the same patient requiring reconstruction. Our group has previously designed such an approach using tissue chambers filled with morcellized bone autograft as a scaffold to autologously generate tissue with a predefined geometry. However, this approach still required donor tissue for filling the tissue chamber. With the recent advances in biodegradable synthetic bone graft materials, it may be possible to minimize this donor tissue by replacing it with synthetic ceramic particles. In addition, these flaps have not previously been transferred to a mandibular defect. In this study, we demonstrate the feasibility of transferring an autologously generated tissue-engineered vascularized bone flap to a mandibular defect in an ovine model, using either morcellized autograft or synthetic bone graft as scaffold material. 01 mei 2015

The existence of multipotent adipose-derived stem cells isolated from human orbital fat (OF) tissue has shown great therapeutic potential in tissue engineering and regenerative medicine. But the use of stem cells for therapeutic applications is influenced by their proliferative and differentiation potentials, which may be affected by the age of the donor. So far there is little knowledge about the effects of donor age on the biological properties of human orbital adipose-derived stem cells (OASCs). The intraorbital fat protrusion in the lower eyelids occurs as an aging process, and the protruded fat is routinely removed during aesthetic surgeries. Based on the ease of OF harvest and the availability of OASCs, we investigated in this study the relationship between age and the differentiation and proliferation potentials of human OASCs. Human orbital adipose samples were harvested from young (with normal lower eyelid appearance) and old donors (having protruded fat pads in the lower eyelids). The morphological properties of orbital adipocytes were assessed and the fat cell size displayed a decreasing trend with advancing age. OASCs were isolated from the fat samples, expanded in vitro and cultured under appropriate inducive conditions. Compared to the young cells, although no difference was found in the cell yield and phenotype expression, aged OASCs showed fewer progenitor cell numbers, reduced proliferative rates, increased senescent features and decreased differentiation potentials towards adipogenic, osteogenic and chondrogenic lineages. Our data suggested that using autologous OASCs from elderly patients for potential therapeutic purposes might be restricted.

Adipose tissue derived stem cells (ASCs) based therapies for the repair and regeneration of various tissues have been widely investigated recently because of their multilineage potential and self-renewal capability. Our previous study demonstrated that autologous ASCs loaded onto natural coral scaffolds could repair cranial critical-sized defects (CSDs) in a canine model. The objective of this study was to determine whether the use of allogeneic ASCs could heal the same defect without the use of immunosuppressive therapy. The pedigree mismatch, mixed lymphocyte reaction assays (MLRs) and allogeneic skin graft experiments were performed to confirm unrelated ASC donors and recipients. A total of 12 adult Beagle dogs were enrolled in this study and divided into two groups. Bilateral cranial CSDs were created in each animal. The right-side defect was treated with allogeneic ASCs delivered onto a coral scaffold, and the left defect was either filled with an autologous ASC/coral composite (Group 1, n = 5) or with one coral scaffold alone (Group 2, n = 5). The systematic immune response and bone healing were evaluated postoperatively. The results showed that allogeneic ASC transplantation did not induce a systemic immune response by the hosts, and allogeneic ASCs could repair the cranial CSDs in an analogous way to that of the autologous cells. Moreover, both the green fluorescently labeled allogeneic and autologous ASCs were detected within the lacunae of newly formed bone in the defect site at 24 weeks, illustrating that the grafted ASCs contributed directly to bone regeneration in vivo. Thus, we concluded that allogeneic ASCs have the capacity to regenerate bone within craniofacial defects, providing an alternative source of seed cells for bone tissue engineering.

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.

As one of the adult stem cells, adipose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs in vitro.The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cooling with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in liquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining and Alizarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured.The survival percentage of the cryopreserved cells was 90.44% +/- 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Alizarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P0.05).Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.

Preservation of tissue-engineered (TE) bone is one of the key problems needed to be solved for its clinic application and industrialization. Traditional cryopreservation has been restricted because of the damages caused by ice formation and solution. Hypothermic preservation at 4 °C has been widely used for the preservation of transplanted organ despite potential negative effects on viability of cells and tissue. 37 °C is the best temperature for maintaining cellular bioactivities. However, 37 °C also has a potential negative effect on preserved cells due to consumption of nutrients and accumulation of by-products. No studies have reported which temperature is more suitable for the preservation of TE bone constructs. The current study explored the feasibility of preservation of TE bone constructs in sealed osteogenic media at 37 °C and 4 °C. Human bone marrow stromal cells (hBMSCs) were seeded into partially demineralized bone matrix (pDBM) scaffolds and cultured for 7 days to form TE bone constructs. The constructs were preserved in sealed osteogenic media at either 37 °C or 4 °C for 5, 7, 9 and 11 days, respectively. Growth kinetics, viability, metabolism and osteogenic capability were evaluated to explore the feasibility of preservation at 37 °C and 4 °C. The constructs cultured in osteogenic media at humidified 37 °C/5%CO2 served as the positive control. The results demonstrated that all the constructs preserved at 4 °C showed negative osteogenic capability at all time points with a much lower level of growth kinetics, viability and metabolism compared to the positive control. However, the constructs preserved at 37 °C showed good osteogenic capability within 7 days with a certain level of growth kinetics, viability and metabolism, although an obvious decrease in osteogenic capability was observed in the constructs preserved at 37 °C over 9 days. These results indicate that the preservation of TE bone constructs is feasible at 37 °C within 7 days in sealed osteogenic media.

Tissue engineering has become a new approach for repairing bone defects. Previous studies indicated that coral scaffolds had been utilized with bone marrow stromal cells (BMSCs) in a variety of approaches for bony reconstruction. In these applications, the degradation rate of the material did not match the rate at which bone was regenerated. In this study, a previously established 30 mm long mandibular segmental defect was repaired with engineered bone using green fluorescent protein-labeled osteogenic BMSCs seeded on porous coral (n = 12). Defects treated with coral alone (n = 12) were used as an experimental control. In the BMSCs/coral group, new bone formation was observed from 4 weeks postoperation, and bony-union was achieved after 32 postoperative weeks. The residual coral volume of the BMSCs/coral grafts at 12 weeks (20-30%) was significantly higher than that at 32 weeks (10-15%, p < 0.05), which was detected by microcomputed tomography and histological examination. The engineered bone with BMSCs/coral achieved satisfactory biomechanical properties at 32 weeks postoperation, which was very close to that of the contralateral edentulous mandible. More importantly, immunostaining demonstrated that the implanted BMSCs differentiated into osteoblast-like cells. In contrast, minimal bone formation with almost solely fibrous connection was observed in the group treated with coral alone. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and biodegradable coral can successfully repair the critical-sized segmental mandibular defects in canines and the seeding cells could be used for bony restoration.

Human adipose-derived stem cells (hASCs) offer great promise for bone tissue engineering because of their osteogenic differentiation potential. At molecular levels, this study investigated the contribution of one of the main members of mitogen-activated protein kinases (MAPKs), extracellular signal-related kinase (ERK), to hASC osteogenic differentiation and the regulation of ERK for the balance between osteogenesis and adipogenesis in hASCs in vitro. As analyzed using western blot, ERK activation in osteo-induced hASCs was initiated at day 7, peaked at day 10, and declined from day 14 to basal levels. As detected using histochemical and biochemical methods, alkaline phosphatase (ALP) activity in hASCs experienced a process similar to that of ERK activation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as revealed by an ALP activity assay, extracellular calcium deposition detection, osteocalcin (OCN) secretion examination, and real-time polymerase chain reaction (PCR) analysis for expression of osteogenesis-relative genes: core binding factor alpha 1, collagen type I, ALP, and OCN. Blockage of ERK phosphorylation in osteo-induced hASCs by PD98059 supplemented with dexamethasone (Dex) led to adipogenic differentiation, as confirmed by Nile Red staining to detect intracellular lipid droplets and real-time PCR analysis for expression of adipogenesis-relative genes: peroxisome proliferator-activated receptor gamma 2 and fatty acid-binding protein. These findings indicated a potential mechanism for the function of ERK in hASC osteogenic differentiation, especially the regulation of ERK in association with Dex for the balance between osteogenesis and adipogenesis, pointing out the significance of ERK signaling pathway for ASCs as a promising cell source for bone tissue engineering.

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

Allogenous demineralized bone matrix (DBM) represents a potential scaffold for bone tissue engineering due to its close relation in structure and function with autologous bone, but its supply is often restricted by donor availability. Thus, an expanded source of human bone is needed. The aim of this study was to evaluate the capacity of partially DBM scaffolds derived from allogenous cancellous bone of osteoporotic femurs to support osteogenesis of human bone marrow stromal cells (BMSCs) in vitro and in vivo in order to assess their potential use in bone tissue-engineering strategies. Human BMSCs of passage 2 were seeded either on osteoporotic bone-derived DBM scaffolds or on normal bone-derived scaffolds and cultured in osteogenic medium for 14 days. To assess the in vitro proliferation potential and osteogenic differentiation of BMSCs on scaffolds, scanning electronic microscopy observation, DNA content assays, and measurements of alkaline phosphatase activity and osteocalcin content were applied; the results displayed no significant differences between the osteoporotic DBM group and the normal DBM group. After 2 weeks of subculture in vitro, the BMSC/DBM composites were subcutaneously implanted into athymic mice for 8 weeks to evaluate their in vivo bone-forming ability. Histological examination showed tissue-engineered bone formation in the DBM pores in both groups, and no significant differences were observed in either the extent or frequency of new bone formation between these two groups. Based on these results, it can be concluded that osteoporotic bone-derived DBM may serve as a promising scaffold for bone tissue engineering.

Tissue engineering techniques have been proven effective in bone regeneration and repairing load-bearing bone defects. Previous studies, however, have heretofore been limited to the use of slowdegradable or natural biomaterials as scaffolds. There are, however, no reports on using biodegradable, synthetic beta-tricalcium phosphate (beta-TCP) as scaffolds to repair weight-bearing bone defects in large animals. In the present study, highly porous beta-TCP scaffolds prepared by the polymeric sponge method were used to repair goat tibial defects. Fifteen goats were randomly assigned to one of three groups, and a 26 mm-long defect at the middle part of the right tibia in each goat was created. In Group A (six goats), a porous beta-TCP ceramic cylinder that had been loaded with osteogenically induced autologous bone marrow stromal cells (BMSCs) was implanted in the defect of each animal. In Group B (six goats), the same beta-TCP ceramic cylinder without any cells loaded was placed in the defect. In Group C (three goats), the defect was left untreated. In Group A, bony union can be observed by gross view, X-ray and micro-computed tomography (Micro-CT) detection, and histological observation at 32 weeks post-implantation. The implanted beta-TCP scaffolds were almost completely replaced by tissue-engineered bone. Bone mineral density in the repaired area of Group A was significantly higher (p0.05) than that of Group B, in which scant new bone was formed in each defect and the beta-TCP hadn't been completely resorbed at 32 weeks. Moreover, the tissue-engineered bone of Group A had similar biomechanical properties as that of the normal left tibia in terms of bending strength and Young's modulus (p0.05). In Group C, little or no new bone was formed, and non-union occurred, showing that the 26 mm segmental defect of the goat tibia was critical sized at 32 weeks. Thus, it can be concluded that the mechanical properties of the BMSCs/beta-TCP composites could be much improved via tissue engineering approach and beta-TCP might be used to repair the weight-bearing segmental defects of goat tibias.

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.