Tissue-engineered Bone Formation Research Articles

Effect of Mechanical Loading on Bone Regeneration in HA/β-TCP/SF Scaffolds Prepared by Low-Temperature 3D Printing In Vivo.

It has been well demonstrated that a dynamic culture environment improves tissue-engineered bone formation in vitro, but little is known about how cyclical mechanical loading induced bone formation in scaffolds in situ. To mimic the organic and inorganic components and multilevel structure of a bony microenvironment, hydroxyapatite/β tricalcium phosphate/silk fibroin(HA/β-TCP/SF) composite scaffolds with macro- and micropores were fabricated in this study. The mechanical properties and structure of the scaffolds were adjusted based on the ratio of organic and inorganic components and three-dimensional (3D) printing parameters. Dynamic sinusoidal loading with different frequencies was applied to the composite scaffold. Mouse bone precursor cells MC3T3-E1 were seeded on the scaffolds, and the cell compatibility of the scaffolds was investigated by MTT, SEM, and HE. The effect of the loading on bone formation in the scaffold in situ was investigated in a rabbit tibia defect model. The scaffold showed viscoelasticity and hysteresis under dynamic sinusoidal loading with different frequencies. With an increase in HA/β-TCP, the stress and modulus of the scaffolds increased. MTT, SEM, and HE results showed that MC3T3-E1 cells could adhere and proliferate on the composite scaffolds. After loading in vivo, the quantity of newly formed bone and the bone volume fraction increased. Micro-CT, undecalcified Van Gieson (VG) staining, and fluorescent double-labeling results suggested that appropriate cyclical mechanical loading at frequencies of 1 and 10 Hz had positive effects on bone formation in situ and it may play a role in clinical bone defect repair.

Understanding tissue-engineered endochondral ossification; towards improved bone formation.

Endochondral ossification (EO) is the process by which the long bones of the body form and has proven to be a promising method in tissue engineering for achieving cell-mediated bone formation. The present review centred on state-of-the-art research pertaining to mesenchymal stem cells (MSCs)-mediated endochondral bone formation, focusing on the role of donor cells, extracellular matrix and host immune cells during tissue-engineered bone formation. Possible research avenues to improve graft outcome and bone output were highlighted, as well as emerging research that, when applied to tissue-engineered bone grafts, offers new promise for improving the likelihood of such grafts transition from bench to bedside.

Platelet-Rich Plasma Pretreatment on Grit-Blasted Titanium Alloy for Enhanced Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

BackgroundAdequate bone formation around titanium alloy implants is integral to successful implantation surgery. Stem cell-coated implants may accelerate peri-implant bone formation. This study investigates the effect of platelet-rich plasma (PRP) pretreatment on a titanium-alloy surface in terms of proliferation and osteogenic differentiation of human adipose-derived stem cells (hADSCs).MethodsAllogenic leukocyte-depleted PRP was obtained from blood supernatants. The hADSCs were isolated from thigh subcutaneous fat tissue. Grit-blasted titanium plugs were used in two different groups. In one group, 200 µL of PRP was added to the grit-blasted titanium plugs. The hADSCs were seeded in two groups: grit-blasted titanium plugs with or without PRP. The number of hADSCs was measured after 4 hours, 3 days, and 7 days of culture using Cell Counting Kit-8. Osteogenesis of hADSCs was measured by using an alkaline phosphatase activity assay on days 7 and 14, and a calcium assay on days 14 and 21. Osteogenic gene expression was measured by using reverse transcription polymerase chain reaction analysis of alkaline phosphatase, osteocalcin, and type I collagen mRNA. The microscopic morphology of grit-blasted titanium plugs with or without PRP was examined with a field-emission scanning electron microscope using a JSM-7401F apparatus on days 1 and 7.ResultsProliferation and osteogenic differentiation of hADSCs were found to be significantly higher on the grit-blasted titanium alloy preprocessed with PRP than the same alloy without pretreatment. Furthermore, a structural fibrillar mesh developed compactly on the grit-blasted titanium alloy with the PRP pretreatment.ConclusionsOur results demonstrate that a hADSC-based approach can be used for tissue-engineered peri-implant bone formation and that PRP pretreatment on the grit-blasted titanium alloy can improve proliferation and osteogenic differentiation of hADSCs.

In Vivo Osteogenic Differentiation of Human Dental Pulp Stem Cells Embedded in an Injectable In Vivo-Forming Hydrogel.

In this study, human dental pulp stem cells (hDPSCs) are examined as a cellular source for bone tissue engineering using an in vivo-forming hydrogel. The hDPSCs are easily harvested in large quantities from extracted teeth. The stemness of harvested hDPSCs indicates their relative tolerance to ex vivo manipulation in culture. The in vitro osteogenic differentiation of hDPSCs is characterized using Alizarin Red S (ARS), von Kossa (VK), and alkaline phosphatase (ALP) staining. The solution of hDPSCs and a methoxy polyethylene glycol-polycaprolactone block copolymer (PC) is easily prepared by simple mixing at room temperature and in no more than 10 s it forms in vivo hydrogels after subcutaneous injection into rats. In vivo osteogenic differentiation of hDPSCs in the in vivo-forming hydrogel is confirmed by micro-computed tomography (CT), histological staining, and gene expression. Micro-CT analysis shows evidence of significant tissue-engineered bone formation in hDPSCs-loaded hydrogel in the presence of osteogenic factors. Differentiated osteoblasts in in vivo-forming hydrogel are identified by ARS and VK staining and are found to exhibit characteristic expression of genes like osteonectin, osteopontin, and osteocalcin. In conclusion, hDPSCs embedded in an in vivo-forming hydrogel may provide benefits as a noninvasive formulation for bone tissue engineering applications.

Quantitative analysis of factors influencing tissue-engineered bone formation by detecting the expression levels of alkaline phosphatase and bone γ-carboxyglutamate protein 2.

Bone tissue engineering is a promising alternative approach that permits the efficient reconstruction of bone defects. There are four elements involved in bone tissue engineering technology, including the seed cells, growth factors, scaffolds and culture environment. The aim of the present study was to evaluate the effect of these factors on bone formation in tissue engineering technology by analyzing the expression of osteogenetic markers using polymerase chain reaction (PCR). Bone marrow mesenchymal stem cells (BMSCs) were extracted from the bone marrow of the bilateral tibial platform of New Zealand white rabbits. In addition, platelet-rich plasma (PRP) samples were prepared from blood extracted from the ear vein of the rabbits. A perfusion bioreactor was used to provide the culture environment, and β-tricalcium phosphate (β-TCP) was used to build the scaffolds. The β-TCP scaffolds were divided into five groups and each group was treated with a different combination of the factors. Next, the composites were implanted into the rabbits. After three months, the expression levels of the new bone formation markers, alkaline phosphatase and bone γ-carboxyglutamate protein 2, were detected using quantitative reverse transcription-PCR analysis. The expression levels of the markers in the experimental groups were higher compared with the negative control group. Comparisons between the experimental groups also revealed statistical significance. Scanning electron microscopy revealed good adhesion and distribution of the BMSCs on the β-TCP scaffold. In conclusion, the PCR results indicated that PRP, BMSCs and the bioreactor exhibited a promoting effect on bone formation.

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery

Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts. Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation. The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells (BMSCs). Methods BMSCs/β-tricalcium phosphate (β-TCP) composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound (LIPUS) treatment group and a control group. These implants were harvested at 5, 10, 25, and 50 days after implantation. The samples were then biomechanically tested and analyzed for alkaline phosphate (ALP) activity and osteocalcin (OCN) content and were also observed by light microscopy. Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group. Histomorphometric analysis revealed a greater degree of soft tissue repair, increased blood flow, better angiogenesis, and more extensive bone formation in the LIPUS groups than in the controls. No significant difference in the compressive strength was found between the two groups. Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β-TCP composites. Chin Med J 2014;127 (4): 669-674

Tissue-Engineered Bone Formation In Vivo for Artificial Laminae of the Vertebral Arch Using β-Tricalcium Phosphate Bioceramics Seeded With Mesenchymal Stem Cells

A rabbit laminectomy model was used to evaluate the efficacy of artificial laminae of vertebral arch using bone marrow-derived mesenchymal stem cells (MSCs) transplanted in porous beta-calcium phosphates (β-TCP) bioceramics. The aim of this study was to establish artificial lamina of the vertebral arch for bone tissue engineering using β-TCP bioceramics seeded with MSCs in a rabbit model of decompressive laminectomy. Decompressive laminectomy may induce various degrees of scar tissue and adhesion formation in the epidural space, and thus is the most common cause of failed back surgery syndrome. However, there is no effective method of bone defect treatment to control and reduce the scar tissue formation. MSCs were harvested from New Zealand rabbits (2-week old) by femoral bone marrow extraction. These cells were seeded into porous β-TCP bioceramics and cultivated for up to 3 weeks in the presence of osteogenic supplements. Segmental defects (20 × 8 mm) were created in 48 adult New Zealand rabbits that underwent laminectomy at the L5 to L6 levels. The animals were transplanted with cell media (control), β-TCP bioceramics (group I), or MSC-loaded β-TCP bioceramics (group II). Bone formation was evaluated after operation using scanning electron microscopy, computed tomography, magnetic resonance imaging, histomorphometry, and immunohistochemistry. Scanning electron microscopy showed that MSCs filled the pores and surfaces of bioceramics in MSC-loaded β-TCP. In addition, significant increases in bone formation were observed in group II compared with other groups. Computed tomography and magnetic resonance imaging at 16 weeks showed that the artificial lamina of the vertebral arch was successfully formed. Hematoxylin-eosin and Masson trichrome staining were used to show the artificial laminae of the vertebral arch and the degraded bioceramics. In addition, immunohistochemistry results showed that the expression of bone morphogenetic protein-2 increased significantly in group II compared with group I at 2,4, and 8 weeks after implantation (P < 0.05). β-TCP bioceramics seeded with MSCs are a promising source of tissue-engineered bone for the artificial lamina of the vertebral arch.

Localized delivery of growth factors for angiogenesis and bone formation in tissue engineering

Angiogenesis is a key component of bone formation. Delivery of growth factors for both angiogenesis and osteogenesis is about to gain important potential as a future therapeutic tool. This review focuses on these growth factors that have dual functions in angiogenesis and osteogenesis, and their localized application. A major hurdle in the clinical development of growth factor therapy so far is how to assure safe and efficacious therapeutic use of such factors and avoid unwanted side effects and toxicity. It is now firmly established from the available information that the type, dose, combinations and delivery kinetics of growth factors all play a decisive role for the success of growth factor therapy. All of these parameters have to be adapted and optimized for each animal model or clinical case. In this review we discuss some important parameters associated with growth factor therapy and present an overview of selected preclinical studies, followed by a conceptual description of both established and proposed delivery strategies meeting therapeutic needs.

Generation of osteogenic construct using periosteal‐derived osteoblasts and polydioxanone/pluronic F127 scaffold with periosteal‐derived CD146 positive endothelial‐like cells

The purpose of this study was to generate tissue-engineered bone using human periosteal-derived osteoblasts (PO) and polydioxanone/pluronic F127 (PDO/pluronic F127) scaffold with preseeded human periosteal-derived CD146 positive endothelial-like cells (PE). PE were purified from the periosteal cell population by cell sorting. One of the important factors to consider in generating tissue-engineered bone using osteoprecursor and endothelial cells and a specific scaffold is whether the function of osteoprecursor and endothelial cells can be maintained in originally different culture medium conditions. After human PE were preseeded into PDO/pluronic F127 scaffold and cultured in endothelial cell basal medium-2 for 7 days, human PO were seeded into the PDO/pluronic F127 scaffold with PE, and then, this cell-scaffold construct was cultured in endothelial cell basal medium-2 with osteogenic induction factors, including ascorbic acid, dexamethasone, and β-glycerophosphate, for a further 7 days. Then, this 2-week cultured construct was grafted into the mandibular defect of miniature pig. Twelve weeks after implantation, the animal was sacrificed. Clinical examination revealed that newly formed bone was seen more clearly in the defect with human PO and PDO/pluronic F127 scaffold with preseeded human PE. The experimental results suggest that tissue-engineered bone formation using human PO and PDO/pluronic F127 scaffold with preseeded human PE can be used to restore skeletal integrity to various bony defects when used in clinics.

Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells

The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.

Small intestine submucosa sponge for in vivo support of tissue-engineered bone formation in the presence of rat bone marrow stem cells

The aim of the current study was to visualize new bone formed in vivo on a small intestine submucosa (SIS) sponge used as a tissue-engineered scaffold for the repair of damaged bone. The SIS sponge provided a three-dimensional pore structure, and supported good attachment and viability of rat bone marrow stem cells (rBMSCs). To examine bone regeneration, we prepared full-thickness bilateral bone defects in the rat crania, and then treated the defects with an implanted SIS sponge or PGA mesh without or with rBMSCs, or left the defects untreated. Bone defects were evaluated by micro-CT and histologically after 2 and 4 weeks. Micro-CT demonstrated a trend toward a decrease in bone void in both the SIS sponge and SIS sponge/rBMSCs groups compared to the control and PGA mesh groups. At 4 weeks, bone formation in defects containing SIS sponge/rBMSCs was significantly greater than in all other groups. A histological analysis after 2 and 4 weeks of implantation showed localized collagen and osteocalcin deposition on SIS sponges and SIS sponges with rBMSCs. These in vivo results indicate that the SIS sponge, implanted at bone-removal defects, facilitated bone regeneration.

In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells in an Injectable In Situ–Forming Gel Scaffold

The sol-to-gel transition occurring at around body temperature makes the MPEG-PCL diblock copolymer an ideal candidate material for use as an injectable in situ-forming gel containing human adipose tissue-derived stem cells (hADSCs). The sol can be prepared at room temperature, and the gel forms at body temperature. Solutions of the copolymer containing hADSCs and osteogenic factors injected into rats formed gel scaffolds at the injection sites. The gels thus formed showed the interconnective pore structure required to support growth, proliferation, and differentiation of hADSCs. Bromodeoxyuridine-labeled hADSCs were confirmed to be present in gels formed in vivo. Bone formation was observed only in gel implants containing both hADSCs and osteogenic factors. Subcutaneous implantation of the in situ-forming gel scaffold demonstrated that hADSCs embedded in the gel stimulated much lower host tissue responses than did the gel alone, probably because of the unique immunomodulatory properties of hADSCs. In conclusion, our data on hADSCs embedded in an in situ gel scaffold suggest that this formulation may provide numerous benefits as a noninvasive alternative for tissue-engineered bone formation.

The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo

A major hurdle to surmount in bone-tissue engineering is ensuring a sufficient oxygen supply to newly forming tissue to avoid cell death or delayed development of osteogenic features. We hypothesized that an oxygen-enriched hydrogel scaffold would enhance tissue-engineered bone formation in vivo. To test this, we used a well-characterized mesenchymal stem cell (MSC) line, Tet-off BMP2 MSC, whose cells were engineered to express recombinant human bone morphogenetic protein-2. Cells were suspended in hydrogel supplemented with perfluorotributylamine (PFTBA) and implanted subcutaneously in an ectopic site, a radial bone defect, or a lumbar paravertebral muscle (mouse model of spinal fusion) in C3H/HeN mice. For controls, we used cells suspended in the same gel without PFTBA. In the ectopic site, there were significant increases in bone formation (2.5-fold increase), cell survival, and osteocalcin activity in the PFTBA-supplemented groups. PFTBA supplementation significantly increased structural parameters of bone in radial bone defects and triggered a significant 1.4-fold increase in bone volume in the spinal fusion model. We conclude that synthetic oxygen carrier supplementation of tissue-engineered implants enhances ectopic bone formation and yields better bone quality and volume in bone-repair and spinal fusion models, probably due to increased cell survival.

Evaluation of Partially Demineralized Osteoporotic Cancellous Bone Matrix Combined with Human Bone Marrow Stromal Cells for Tissue Engineering: An In Vitro and In Vivo Study

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.

Stem Cells from Human Fat as Cellular Delivery Vehicles in an Athymic Rat Posterolateral Spine Fusion Model

Mesenchymal stem cells derived from human liposuction aspirates, termed processed lipoaspirate cells, have been utilized as cellular delivery vehicles for the induction of bone formation in tissue engineering and gene therapy strategies. In this study, we sought to evaluate the efficacy of bone morphogenetic protein (BMP)-2-producing adipose-derived stem cells in inducing a posterolateral spine fusion in an athymic rat model. Single-level (L4-L5) intertransverse spinal arthrodesis was attempted with use of a type-I collagen matrix in five groups of athymic rats, with eight animals in each group. Group I was treated with 5 x 10(6) adipose-derived stem cells transduced with an adenoviral vector containing the BMP-2 gene; group II, with 5 x 10(6) adipose-derived stem cells treated with osteogenic media and 1 microg/mL of recombinant BMP-2 (rhBMP-2); group III, with 10 microg of rhBMP-2; group IV, with 1 microg of rhBMP-2; and group V, with 5 x 10(6) adipose-derived stem cells alone. The animals that showed radiographic evidence of healing were killed four weeks after cell implantation and were examined with plain radiographs, manual palpation, microcomputed tomography scanning, and histological analysis. All eight animals in group I demonstrated successful spinal fusion, with a large fusion mass, four weeks postoperatively. Furthermore, group-I specimens consistently revealed spinal fusion at the cephalad level (L3 and L4), where no fusion bed had been prepared surgically. In contrast, despite substantial BMP-2 production measured in vitro, group-II animals demonstrated minimal bone formation even eight weeks after implantation. Of the groups treated with the application of rhBMP-2 alone, the one that received a relatively high dose (group III) had a higher rate of fusion (seen in all eight specimens) than the one that received the low dose (group IV, in which fusion was seen in four of the eight specimens). None of the group-V animals (treated with adipose-derived stem cells alone) demonstrated successful spine fusion eight weeks after the surgery. Adipose-derived stem cells show promise as gene transduction targets for inducing bone formation to enhance spinal fusion in biologically stringent environments.

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me 2SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.

Tissue-engineered bone formation using human bone marrow stromal cells and novel β-tricalcium phosphate**This work was supported by the National Basic Research Program of China (G1999054300, 2005CB522700) and Shanghai Science and Technology Development Foundation.

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel β-tricalcium phosphate (β-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous β-TCP scaffolds with pores of 300–500 µm in size were prepared by the polymeric sponge method. β-TCP scaffolds with the dimension of 3 mm × 3 mm × 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 (−) (p < 0.05). These results indicated that the novel porous β-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 β-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 β-TCP scaffolds (−) at each time point checked. The above findings illustrate that the novel porous β-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

Enhancement of tissue engineered bone formation by a low pressure system improving cell seeding and medium perfusion into a porous scaffold

To obtain more extensive bone formation in composites of porous ceramics and bone marrow stromal cells (BMSCs), we hypothesized that a low-pressure system would serve to facilitate the perfusion of larger number of BMSCs into the porous scaffold, enhancing bone formation within the composites. After culturing BMSCs in osteogenic medium, porous blocks of β-tricalcium phosphate (β-TCP) were soaked in the cell suspension. Composites of the block and BMSCs were put immediately into a vacuum desiccator. Low pressure was applied to the low pressure group, while controls were left at atmospheric pressure. Composites were incubated in vitro or subcutaneously implanted into syngeneic rats, then analyzed biologically and histologically. In the in vitro group, cell suspension volume, cell seeding efficiency, alkaline phosphatase (ALP) activity, and DNA content in the β-TCP blocks were significantly higher in low pressure group than in the controls. Scanning electron microscopy (SEM) demonstrated that a greater number of cells covered the central parts of the composites in the low pressure group. ALP activity in the composites was increased at 3 and 6 weeks after implantation into rats. Histomorphometric analysis revealed more uniform and extensive bone formation in the low pressure group than in the controls. The application of low pressure during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue-engineered bone formation.

A comparative qualitative histological analysis of tissue-engineered bone using bone marrow mesenchymal stem cells, alveolar bone cells, and periosteal cells

For tissue-engineered bone formation, autogenous osteogenic cells are of paramount importance for successful bone formation. In order to investigate the donor cell-related differences in tissue-engineered bone, cultured bone marrow mesenchymal stem cells, cultured alveolar bone cells, and cultured periosteal cells were examined for their in vivo potential to form bone. These cells were isolated from dogs, expanded in vitro, mixed with autologous fibrin glue and BMP-2, and then injected into the subcutaneous space on the dorsum of nude mice. Bone formation was evaluated at 12 weeks. Histomorphometric analysis demonstrated that the subcutaneous nodules formed in nude mice contained 26.9% newly formed bone when using the bone marrow mesenchymal stem cells, 41.1% newly formed bone when using the alveolar bone cells, and 58.2% newly formed bone when using the periosteal cells. The results suggest that periosteal cells are the best choice for enhancing bone formation in tissue engineering of bone regeneration.

Tissue Engineered Bone Formation with Polymer/Ceramic Composites by Press-and-Baking Method

A novel process was developed to fabricate polymer/ceramic composites for bone tissue engineering. The mixture of polylactic acid (PLA), calcium metaphosphate (CMP), and NaCl were compressed and subsequently heated. After dissolving the NaCl salts, porous biodegradable polymer/ceramic composite scaffolds were formed. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting method, in terms of pore structure, pore size distribution, and mechanical properties. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. Cells were better grown to form tissue-like structures on CMP/PLA composites fabricated by the Press-and-Baking method. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by Press-and-Baking compared to them by solvent casting. Taken together, these results suggest that CMP promote cell differentiation and proliferation via direct interaction with cells in the CMP/PLA composites. This novel PLA/CMP composite will be applicable for bone tissue engineering to support and cell differentiation and growth.