β-TCP Scaffolds Research Articles

Effects of formaldehyde solution and nanoparticles on mechanical properties and biodegradation of gelatin/nano β-TCP scaffolds

In this study, gelatin/beta tricalcium phosphate (β-TCP) nanocomposite scaffolds were prepared by solvent casting method. The cross-linking method was carried out by adding formaldehyde to gelatin. The microparticles of sodium chloride were used as porogen agent. Characterization of nano β-TCP was performed using XRD, FTIR, and SEM. Results showed that the size of the particles is about 100 nm with spherical morphology. In addition, the scaffold characterization was carried out using FTIR and SEM techniques. Observations showed a porous texture with pore size between 100 and 400 μm. The biodegradability and bioactivity evaluations of the scaffolds were done by immersing them in a simulated body fluid solution for different time periods. The biodegradability studies demonstrated a reduction in the degradation rate of gelatin/β-TCP nanocomposite scaffolds due to the presence of β-TCP nanoparticles. The obtained results of bioactivity tests confirmed the formation of apatite layer on the surface of the scaffolds. Furthermore, the effects of porosity, cross-linking agent, and β-TCP nanoparticles on the bending and compressive properties of the composite scaffolds were examined. According to the mechanical examinations of the scaffolds, the best bending and compressive properties occurred in the presence of 10 and 20 wt% of β-TCP nanoparticles, respectively. The appropriate mechanical properties and biodegradation rate for tissue engineering applications obtained at 1 g of the formaldehyde solution.

The effect of rhBMP-2 and PRP delivery by biodegradable β-tricalcium phosphate scaffolds on new bone formation in a non-through rabbit cranial defect model

This study evaluated whether the combination of biodegradable β-tricalcium phosphate (β-TCP) scaffolds with recombinant human bone morphogenetic protein-2 (rhBMP-2) or platelet-rich plasma (PRP) could accelerate bone formation and increase bone height using a rabbit non-through cranial bone defect model. Four non-through cylindrical bone defects with a diameter of 8-mm were surgically created on the cranium of rabbits. β-TCP scaffolds in the presence and absence of impregnated rhBMP-2 or PRP were placed into the defects. At 8 and 16 weeks after implantation, samples were dissected and fixed for analysis by microcomputed tomography and histology. Only defects with rhBMP-2 impregnated β-TCP scaffolds showed significantly enhanced bone formation compared to non-impregnated β-TCP scaffolds (P < 0.05). Although new bone was higher than adjacent bone at 8 weeks after implantation, vertical bone augmentation was not observed at 16 weeks after implantation, probably due to scaffold resorption occurring concurrently with new bone formation.

The role of miR-31-modified adipose tissue-derived stem cells in repairing rat critical-sized calvarial defects

With the increasing application of microRNAs (miRNAs) in the treatment and monitoring of different diseases, miRNAs have become an important tool in biological and medical research. Recent studies have proven that miRNAs are involved in the osteogenic differentiation of stem cells. However, few studies have reported the use of miRNA-modified adult stem cells to repair critical-sized defects (CSDs) using tissue engineering technology. It is known that miR-31 is a pleiotropically acting miRNA that inhibits cancer metastasis and targets special AT-rich sequence-binding protein 2 (Satb2) in fibroblasts. However, it is not clear whether the function of miR-31 is to enhance adipose tissue-derived stem cell (ASC) osteogenesis, along with its association with Satb2, during osteogenic differentiation and bone regeneration. In this study, we systematically evaluated the function of miR-31 in enhancing ASC osteogenesis and the therapeutic potential of miR-31-modified ASCs in a rat CSD model with β-tricalcium phosphate (β-TCP) scaffolds. ASCs were treated with lentivirus (Lenti)-miR-31, Lenti-as-miR-31 (antisense) or Lenti-NC (negative control). These genetically modified ASCs were then combined with β-TCP scaffolds to repair CSDs in rats. The results showed that in cultured ASCs in vitro, Lenti-as-miR-31 significantly enhanced osteogenic mRNA and protein expression when compared with the Lenti-NC group. Moreover, we firstly found that a Runt-related transcription factor 2 (Runx2), Satb2 and miR-31 regulatory loop triggered by bone morphogenetic protein-2 (BMP-2) plays an important role in ASCs' osteogenic differentiation and bone regeneration. More importantly, we found that miR-31-knockdown ASCs dramatically improved the repair of CSDs, including increased bone volume, increased bone mineral density (BMD) and decreased scaffold residue in vivo. These data confirm the essential role of miR-31-modified ASCs in osteogenesis in vitro and in vivo.

Orbital wall repair in canines with beta‐tricalcium phosphate and induced bone marrow stromal cells

Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to evaluate prefabricated beta-tricalcium phosphate (β-TCP) combined with autologous bone marrow stromal cells (BMSCs) to repair orbital wall defect in canine models. Defects measuring 10 mm in diameter were created in the orbital medial walls of 12 dogs. The orbits were randomly divided into five groups: group 1, repaired with osteogenesis-induced BMSCs/TCP constructs; group 2, repaired with noninduced BMSCs/TCP constructs; group 3, repaired with β-TCP scaffolds only; group 4, normal group; group 5, negative control (bone defect without treatment). Computed tomography (CT) scanning, gross observation, bone density measurements, micro-CT, and histological observations were performed. In group 1, new bone was observed with only a small amount of residual material, and bony union was achieved 3 months after surgery. In contrast, the constructs showed slow degradation with minimal bone formation in groups 2 and 3. Furthermore, the appearance and bone density of the constructs in group 1 were similar to that of normal bone: the constructs were covered with complete mucosa, and new alveolate plate grew into the ethmoidal sinuses. A large bone defect remained in group 5. This study demonstrated that biologic scaffolds composed of β-TCP and osteogenesis-induced BMSCs have been successfully used to restore bone functionality in animal models, which may provide a potential clinical approach for orbital wall repair and bone regeneration in humans.

Reinforcing bioceramic scaffolds with in situ synthesized ε‐polycaprolactone coatings

In situ ring-opening polymerization of ε-caprolactone (ε-CL) was performed to coat β-tricalcium phosphate (β-TCP) scaffolds fabricated by robocasting in order to enhance their mechanical performance while preserving the predesigned macropore architecture. Concentrated colloidal inks prepared from β-TCP commercial powders were used to fabricate porous structures consisting of a three-dimensional mesh of interpenetrating rods. Then, ε-CL was in situ polymerized within the ceramic structure using a lipase as catalyst and toluene as solvent, to obtain a highly homogeneous coating and full impregnation of in-rod microporosity. The strength and toughness of scaffolds coated by ε-polycaprolactone (ε-PCL) were significantly increased (twofold and fivefold increase, respectively) over those of the bare structures. Enhancement of both properties is associated to the healing of preexisting microdefects in the bioceramic rods. These enhancements are compared to results from previous work on fully impregnated structures. The implications of the results for the optimization of the mechanical and biological performance of scaffolds for bone tissue engineering applications are discussed.

Thein vitroandin vivocementogenesis of CaMgSi2O6bioceramic scaffolds

The goal of periodontal tissue engineering is to regenerate alveolar bone, root cementum and periodontal ligament. To achieve this goal, bioactive scaffolds play an important role in inducing in vitro osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) and in vivo bone/cementum formation. Diopside (DIOP: CaMgSi2O6) ceramics have shown excellent in vitro bioactivity for potential bone repair application. However, there is no study about DIOP porous scaffolds for periodontal tissue engineering. The aim of this study is to prepare DIOP scaffolds and investigate their in vitro and in vivo osteogenesis/cementogenesis for periodontal regeneration application. DIOP scaffolds with highly porous architecture were prepared and β-tricalcium phosphate (β-TCP) scaffolds were used for the control. The interaction of DIOP scaffolds with PDLCs was studied by investigating cell attachment, proliferation and ostegenic/cementogenic differentiation of PDLCs. DIOP scaffolds were implanted into the periodontal defects of beagle dogs to evaluate their in vivo osteogenesis/cementogenesis by hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase staining, and immunohistochemistry (type I collagen: Col I; cementum attachment protein) analyses. The results have shown that DIOP scaffolds supported the attachment and proliferation of PDLCs. DIOP scaffolds significantly enhanced osteogenesis/cementogenesis-related gene expression (Col 1, Runx2, transforming growth factor beta 1, and bone morphogenetic protein 2) of PDLCs, compared to β-TCP scaffolds. The in vivo study showed that DIOP scaffolds induced new bone and cementum regeneration of periodontal tissue defects. The rate of new bone and cementum in DIOP scaffolds is comparable to that in conventional β-TCP scaffolds. Our results indicated that silicate-based DIOP ceramics could not only be used for bone tissue engineering, but also for periodontal tissue engineering due to their excellent in vitro and in vivo osteogeneis/cementogenesis.

Impregnation of β‐tricalcium phosphate robocast scaffolds by in situ polymerization

Ring-opening polymerization of ε-caprolactone (ε-CL) and L-lactide (LLA) was performed to impregnate β-tricalcium phosphate (β-TCP) scaffolds fabricated by robocasting. Concentrated colloidal inks prepared from β-TCP commercial powders were used to fabricate porous structures consisting of a 3D mesh of interpenetrating rods. ε-CL and LLA were in situ polymerized within the ceramic structure by using a lipase and stannous octanoate, respectively, as catalysts. The results show that both the macropores inside the ceramic mesh and the micropores within the ceramic rods are full of polymer in either case. The mechanical properties of scaffolds impregnated by in situ polymerization (ISP) are significantly increased over those of the bare structures, exhibiting similar values than those obtained by other, more aggressive, impregnation methods such as melt-immersion (MI). ISP using enzymatic catalysts requires a reduced processing temperature which could facilitate the incorporation of growth factors and other drugs into the polymer composition, thus enhancing the bioactivity of the composite scaffold. The implications of these results for the optimization of the mechanical and biological performance of scaffolds for bone tissue engineering applications are discussed.

Microporous calcium phosphate ceramics as tissue engineering scaffolds for the repair of osteochondral defects: Histological results

Treatment of defects in joint cartilage aims to re-establish normal joint function. In vitro experiments have shown that the application of synthetic scaffolds is a promising alternative to existing therapeutic options. A sheep study was conducted to test the suitability of microporous pure β-tricalcium phosphate (TCP) ceramics as tissue engineering scaffolds for the repair of osteochondral defects. Cylindrical plugs of microporous β-TCP (diameter: 7mm; length: 25mm; porosity: 43.5±2.4%; pore diameter: ∼5μm) with interconnecting pores were used. Scaffolds were seeded with autologous chondrocytes in vitro and cultured for 4weeks. A drill hole (diameter 7mm) was placed in both medial femoral condyles of sheep. For the left knee the defect was filled with a TCP plug and for the right knee the defect was left empty. After 6, 12, 26 and 52weeks, seven animals from each group were killed and studied. The samples were examined employing histological, histomorphometric and immunohistological methods as well as various imaging techniques (X-ray, microcomputer tomography and scanning electron microscopy). After explantation the cartilage defects were first assessed macroscopically. There were no signs of infection or inflammation. Histological grading scales were used for assessment of bony integration and cartilage repair. An increasing degradation (81% after 52weeks) of the ceramic with concomitant bone formation was observed. The original structure of cancellous bone was almost completely restored. After 26 and 52weeks, collagen II-positive hyaline cartilage was detected in several samples. New subchondral bone had formed. The formation of cartilage began at the outer edge and proceeded to the middle. According to the O’Driscoll score, values corresponding to healthy cartilage were not reached after 1year. Integration of the newly formed cartilage tissue into the surrounding native cartilage was found. The formation of biomechanical stable cartilage began at the edge and progressed towards the centre of the defect. After 1year this process was still not completed. Microporous β-TCP scaffolds seeded with chondrocytes are suitable for the treatment of osteochondral defects.

Correlation between properties and microstructure of laser sintered porous β-tricalcium phosphate bone scaffolds

A porous β-tricalcium phosphate (β-TCP) bioceramic scaffold was successfully prepared with our homemade selective laser sintering system. Microstructure observation by a scanning electron microscope showed that the grains grew from 0.21 to 1.32 μm with the decrease of laser scanning speed from 250 to 50 mm min−1. The mechanical properties increased mainly due to the improved apparent density when the laser scanning speed decreased to 150 mm min−1. When the scanning speed was further decreased, the grain size became larger and the mechanical properties severely decreased. The highest Vickers hardness and fracture toughness of the scaffold were 3.59 GPa and 1.16 MPa m1/2, respectively, when laser power was 11 W, spot size was 1 mm in diameter, layer thickness was 0.1–0.2 mm and laser scanning speed was 150 mm min−1. The biocompatibility of these scaffolds was assessed in vitro with MG63 osteoblast-like cells and human bone marrow mesenchymal stem cells. The results showed that all the prepared scaffolds are suitable for cell attachment and differentiation. Moreover, the smaller the grain size, the better the cell biocompatibility. The porous scaffold with a grain size of 0.71 μm was immersed in a simulated body fluid for different days to assess the bioactivity. The surface of the scaffold was covered by a bone-like apatite layer, which indicated that the β-TCP scaffold possesses good bioactivity. These discoveries demonstrated the evolution rule between grain microstructure and the properties that give a useful reference for the fabrication of β-TCP bone scaffolds.

Expression of caveolin-1 in the early phase of beta-TCP implanted in dog mandible

Caveolin is an essential and signature protein of caveolae. Caveolin-1 participates in signal transduction processes by acting as a scaffolding protein that concentrates, organizes and functional regulates signalling molecules within caveolar membranes. Beta-tricalcium phosphate (β-TCP) has been widely used for scaffold in tissue engineering due to its high biodegradability, osteoconductivity, easy manipulation, and lack of histotoxicity. To better understand the role of caveolin-1 in bone homeostasis and response to β-TCP scaffold, β-TCP was implanted into the dog mandible defects in beagle dogs, and gene expression profiles were examined focused on the molecular components involved in caveolin-1 regulation. Here we showed the quantitative imageology analysis characterized using in vivo micro-computed tomography (CT) images at 4 and 7 days after β-TCP implanted in dog mandibles. The bone reformation by using the β-TCP scaffolds began within 4 days of surgery, and was healing well at 7 days after surgery. Higher mRNA level of caveolin-1 was observed in β-TCP-implanted Beagle dog mandibles compared with controls at day 4 and day 7 post-surgery. The enhancement of caveolin-1 by β-TCP was further confirmed by immunohistochemistry and immunofluorescence analysis. We further revealed increased Smad7 and Phospho Stat3 expression in β-TCP-implanted specimens. Taken together, these results suggest that the enhancement of caveolin-1 play an important role in accelerating bone formation by β-TCP.

&lt;i&gt;In Vivo&lt;/i&gt; Osteogenic Effect of Porous β- CaSiO&lt;sub&gt;3&lt;/sub&gt;/PDLGA Composite Scaffolds

The aim of the present study is to evaluate the in vivo biological behaviour of porous β-CS/PDLGA scaffolds. The scaffolds were implanted in critical-sized femur defects ( 6 ×10 mm) for 4, 12 and 20 weeks with β-TCP scaffolds as the control. The in vivo bone regeneration of the scaffolds were investigated using sequential histological evaluations and Micro-CT technology. Results showed that the β-CS/PDLGA scaffolds could stimulate bone regeneration and degrade progressively at a rate proportionate with the regeneration of new bone as compared with β-TCP scaffolds. The present study suggested the potential application of β-CS/PDLGA scaffolds in hard tissue regeneration.

GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases

Background:The current management of large mandibular resection defects involves harvesting of autogenous bone grafts and repeated bending of generic reconstruction plates. However, the major disadvantage of harvesting large autogenous bone grafts is donor site morbidity and the major drawback of repeated reconstruction plate bending is plate fracture and difficulty in reproducing complex facial contours. The aim of this study was to describe reconstruction of three mandibular ameloblastoma resection defects using tissue engineered constructs of beta-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (rhBMP-2), and Good Manufacturing Practice (GMP) level autologous adipose stem cells (ASCs) with progressively increasing usage of computer-aided manufacturing (CAM) technology.Materials and Methods:Patients’ three-dimensional (3D) images were used in three consecutive patients to plan and reverse-engineer patient-specific saw guides and reconstruction plates using computer-aided additive manufacturing. Adipose tissue was harvested from the anterior abdominal walls of three patients before resection. ASCs were expanded ex vivo over 3 weeks and seeded onto a β-TCP scaffold with rhBMP-2. Constructs were implanted into patient resection defects together with rapid prototyped reconstruction plates.Results:All three cases used one step in situ bone formation without the need for an ectopic bone formation step or vascularized flaps. In two of the three patients, dental implants were placed 10 and 14 months following reconstruction, allowing harvesting of bone cores from the regenerated mandibular defects. Histological examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed.Discussion:Constructs with ASCs, β-TCP scaffolds, and rhBMP-2 can be used to reconstruct a variety of large mandibular defects, together with rapid prototyped reconstruction hardware which supports placement of dental implants.

Porous nagelschmidtite bioceramic scaffolds with improved in vitro and in vivo cementogenesis for periodontal tissue engineering

Bioactive scaffolds play an important role in periodontal tissue engineering. Nagelschmidtite (NAGEL:Ca7P2Si2O16) is a newly synthesized bioceramic which can stimulate the proliferation and osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for potential periodontal regeneration. The aim of this study is to prepare porous NAGEL scaffolds and to systematically investigate their in vitro and in vivo osteogenesis/cementogenesis. The NAGEL scaffold with highly porous structure and large pores was successfully prepared by a spongy-templated method. The interaction of the NAGEL scaffold with PDLCs was studied by investigating cell attachment, proliferation and osteogenic differentiation of PDLCs. NAGEL scaffolds were implanted into the periodontal defects of beagle dogs to evaluate their in vivo osteogenesis/cementogenesis. The results showed that NAGEL scaffolds supported the attachment and proliferation of PDLCs, and significantly enhanced osteogenesis/cementogenesis-related gene expression of PDLCs, compared to β-TCP scaffolds. The in vivo study showed that the rate of new bone formation in NAGEL scaffolds was higher than that in conventional β-TCP scaffolds, which was evidenced by the significantly improved BSP and OPN expression at both 4 and 8 weeks. The results suggested that NAGEL scaffolds could be used for periodontal tissue engineering with significantly improved in vitro and in vivo osteogenesis/cementogenesis.

In silico Approaches for the Identification of Optimal Culture Condition for Tissue Engineered Bone Substitutes

Tissue engineered bone grafts are among the most promising approaches to heal large bone defects. An in vitro culture phase prior to transplantation provides the opportunity to optimize the graft properties. In our study we developed computational models for the investigation of the deformation, perfusion and revascularization of a porous β-tricalcium phosphate (β-TCP) scaffold seeded with human bone marrow derived mesenchymal stem cells (MSCs). The deformation model allows predicting the resulting forces in the β-TCP scaffold due to different external loadings. In addition to pressure related forces, fluid convection within the scaffold also exerts shear stress onto the cells. However, perfusion is necessary for the supply of the cells with nutrients such as glucose. To find an optimal ratio between shear stress and nutrients supply, we develop fluidic models for the β-TCP scaffold. Furthermore to the scaffold specifications, also the cellular glucose consumption of osteogenic differentiated and undifferentiated MSCs was integrated in the computational model. Beyond the in vitro culture phase, bone grafts have to be supplied by the host ’ s vascular system. Therefore angiogenesis has to be induced, e.g. by preloading the graft with pro angiogenic factors such as VEGF-A. A stochastic model, based on the Fokker-Planck equation was developed to investigate the impact of a given cytokine gradient onto the endothelial cell migration. The stochastic model was parameterized by data derived from live cell imaging studies. Keywords: Tissue engineering, Modelling, Simulation, MSC, Flow, Bone, Young's modulus, Scaffold, Diffusion, Metabolic rates

In silico Approaches for the Identification of Optimal Culture Condition for Tissue Engineered Bone Substitutes

Tissue engineered bone grafts are among the most promising approaches to heal large bone defects. An in vitro culture phase prior to transplantation provides the opportunity to optimize the graft properties. In our study we developed computational models for the investigation of the deformation, perfusion and revascularization of a porous β-tricalcium phosphate (β-TCP) scaffold seeded with human bone marrow derived mesenchymal stem cells (MSCs). The deformation model allows predicting the resulting forces in the β-TCP scaffold due to different external loadings. In addition to pressure related forces, fluid convection within the scaffold also exerts shear stress onto the cells. However, perfusion is necessary for the supply of the cells with nutrients such as glucose. To find an optimal ratio between shear stress and nutrients supply, we develop fluidic models for the β-TCP scaffold. Furthermore to the scaffold specifications, also the cellular glucose consumption of osteogenic differentiated and undifferentiated MSCs was integrated in the computational model. Beyond the in vitro culture phase, bone grafts have to be supplied by the host ’ s vascular system. Therefore angiogenesis has to be induced, e.g. by preloading the graft with pro angiogenic factors such as VEGF-A. A stochastic model, based on the Fokker-Planck equation was developed to investigate the impact of a given cytokine gradient onto the endothelial cell migration. The stochastic model was parameterized by data derived from live cell imaging studies.

β-TCP Scaffolds with an Interconnected and Aligned Porosity Fabricated via Ice-Templating

Pure β-TCP scaffolds with an aligned, lamellar, open and interconnected porosity were fabricated by the ice-templating process. The morphology of the scaffold was analysed and the mechanical properties of the different scaffolds were tested by compression tests. The structural sizes of the scaffolds (pore width and ceramic cell wall thickness) were adjusted by the onset of a constant ice front velocity. For this purpose a freezing device was developed and a specific solution of the non-steady heat equation with a disturbing factor (phase transformation energy from water) was used to model the process parameters. It was found that increasing the ice front velocity decreases the structural sizes and consequently increases the compression strength of the scaffold.

Influence of Architecture of β-Tricalcium Phosphate Scaffolds on Biological Performance in Repairing Segmental Bone Defects

BackgroundAlthough three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolds serve as promising bone graft substitutes for the segmental bone defect treatment, no consensus has been achieved regarding their optimal 3D architecture.MethodsIn this study, we has systematically compared four types of β-TCP bone graft substitutes with different 3D architectures, including two types of porous scaffolds, one type of tubular scaffolds and one type of solid scaffolds, for their efficacy in treating segmental bone defect in a rabbit model.ResultsOur study has demonstrated that when compared to the traditional porous and solid scaffolds, tubular scaffolds promoted significantly higher amount of new bone formation in the defect regions as shown by X-ray, micro CT examinations and histological analysis, restored much greater mechanical properties of the damaged bone evidenced by the biomechanical testing, and eventually achieved the complete union of segmental defect. Moreover, the implantation of tubular scaffolds enhanced the neo-vascularization at the defect region with higher bone metabolic activities than others, as indicated by the bone scintigraphy assay.ConclusionsThis study has further the current knowledge regarding the profound influence of overall 3D architecture of β-TCP scaffolds on their in vivo defect healing performance and illuminated the promising potential use of tubular scaffolds as effective bone graft substitute in treating large segmental bone defects.

The influence of prefreezing temperature on pore structure in freeze-dried β-TCP scaffolds

A combined method of tricalcium phosphate (TCP) scaffold production, which comprised negative mold and scaffold fabrication, was reported in this study. The negative mold structure was designed by computer and fabricated by fused deposition modeling (FDM) technology, while the TCP scaffold was produced by freeze-drying technology under different prefreezing temperatures of -10 degrees C, -30 degrees C, and -86 degrees C and thermal treatment to get beta-TCP. The scaffold structure was evaluated with X-ray, scanning electron microscopy (SEM), compressive mechanical testing, and micro-computerized tomography (micro-CT). The cell-scaffold interaction was studied by culturing dog bone marrow stromal cells (BMSCs) on the scaffolds and assessing differentiated BMSC function by measuring cellular alkaline phosphatase (ALP) activity. The results showed good interconnectivity and good pore distribution with the pore size ranging from 50 to 250 microm and compressive modulus of 1.18 MPa at a prefreezing temperatures of -10 degrees C. In vitro cell culture results indicated that the porous scaffolds were not toxic to bone cells. These results demonstrate that rapid prototyping and freeze-drying technologies for creating beta-TCP scaffolds are promising for bone tissue engineering.

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable β-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7days, but HUVECs reached similar levels of proliferation after 14days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.

In situ formation of nano-hydroxyapatite whisker reinfoced porous β-TCP scaffolds

Graphical abstractDisplay Omitted Highlights? Nano-HA whiskers were born in the porous β-TCP ceramic during in situ synthesis. ? The thermal stability of nano-HA whiskers improved significantly. ? The porous nHAW/β-TCP scaffolds exhibited higher mechanical properties. ? The nano-HA whisker could improve cell adhesion and proliferation on the scaffolds. The preparation of porous ceramic scaffolds with nano-hydroxyapatite (HA) whiskers was reported in this work. By adding kalium ion and adjusting the pH value of solution, the thermal stability of nano-HA whiskers were improved significantly. The whiskers in the ceramic scaffolds can be kept completely after sintered at 1000?C. The nano-HA whisker was about 2µm in length and 100nm in diameter. The raw powders and scaffolds were observed by SEM and TEM, and the phase composition of samples was determined by XRD. The nHAW/β-TCP/BG scaffolds exhibited interconnected porous structures and enhanced mechanical properties as compared to those β-TCP/BG scaffolds. In order to assess the efforts of nano-HA whiskers on the behavior of osteoblasts cultured in vitro, the scaffolds were seeded with MC3T3-E1 and cultured for the same period. The cell viability and proliferation was assessed by the MTT assay and observed using laser scanning confocal microscopy. The Results suggest that the nano-HA whisker could be used to improve the cell adhesion and proliferation.