Goat Bone Marrow Stromal Cells Research Articles

Synthesis and Characterization of Hydroxyapatite/chitosan Composite Material for Medical Enginering Applications

This study is to synthesize the HA-Chitosan composite materials for medical enginerging applications. XRD and SEM analyses confirmed the precipitation and dispersion of HA on the structural network of chitosan polymer material. The dispersion of HA particles depended on the concentration of the chitosan solution. The volumetric mass density and Compressive strength of composite HA-Chtosan were also determined. These values increased as a function of modeling press. In addition, volumetric mass density and compressive strength in composite containing higher chitosan had higher values.
 Keywords
 Hydroxyapatite (HA), chitosan, characterization, modeling press, compressive strength
 References
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Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil.

To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: The effect of silanol groups

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si–OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si–OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL–Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL–Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL–Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL–Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.

The effect of Arbas Cashmere goat bone marrow stromal cells on production of transgenic cloned embryos

The aim of this study was to develop a method for the in vitro separation and culture of Arbas Cashmere goat bone marrow stromal cells (gBMSCs). Arbas Cashmere gBMSCs were isolated and cultured in vitro, and cell surface markers were identified immunohistochemically. The gBMSCs were differentiated into neurocytes and osteoblasts, and the expression of neuron-specific enolase and osteocalcin was identified by immunohistochemistry. The gBMSCs and goat fetal fibroblast cells (gFFCs) were compared for transient transfection efficiency and fluorescent colony-forming efficiency with Arbas Cashmere gFFCs as a control. pDsRed2-1 encodes DsRed2, a variant of the Discosoma sp. red fluorescent protein (DsRed). In addition, the coding sequence for DsRed2 contains a series of silent base-pair changes for higher expression in mammalian cells. Of the gBMSCs-pDsRed2-1, one fraction was tested for pluripotency, whereas the other fraction was manipulated using somatic cell nuclear transfer, and the in vitro growth status of transgenic embryos derived from gBMSCs-pDsRed2-1 and gFFCs-pDsRed2-1 was compared. The findings showed that gBMSCs were isolated and amplified to express CD29, CD44, and CD90 through adherent culture, with no marked signs of aging after multiple passages. Expression of neuron-specific enolase and osteocalcin by gBMSCs and gBMSCs-pDsRed2-1 was strongly induced by neuronal and osteogenic differentiation, whereas the integrated exogenous genes did not influence pluripotency (P > 0.05). The transient transfection efficiencies of gBMSCs and gFFCs after 48 hours were not significantly different; however, the fluorescent colony-forming efficiency of gBMSCs-pDsRed2-1 after G418 screening was approximately 13% higher than that of gFFCs-pDsRed2-1. The convergence and cleavage rates of cloned embryos derived from gBMSCs-pDsRed2-1 were higher than those derived from gFFCs-pDsRed2-1, whereas their eight-cell and blastocyst rates were similar. The red fluorescent protein expression levels were higher in transgenic embryos derived from gBMSCs-pDsRed2-1 compared with those derived from gFFCs-pDsRed2-1 (48.8% vs. 31.1%, respectively) (P < 0.01). Real-time quantitative Polymerase Chain Reaction analysis showed that DsRed2-1 messenger RNA expression of cloned embryos derived from gBMSCs was 2.24 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). Similarly, Western blot analysis showed that DsRed2 protein expression of cloned embryos derived from gBMSCs-pDsRed2-1 was 1.29 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). In this study, gBMSCs were also used for somatic cell nuclear transfer and shown to provide effective nuclear donor cells for breeding new genetically modified varieties of livestock.

Assessing the repair of critical size bone defects performed in a goat tibia model using tissue-engineered constructs cultured in a bidirectional flow perfusion bioreactor

This work evaluated invivo performance of a tissue-engineered bone-like matrix obtained by culturing cell-scaffold constructs in a flow perfusion bioreactor, designed to enable culture of large constructs, envisioning the regeneration of critical-sized defects. A blend of starch with polycaprolactone scaffolds was seeded with goat bone marrow stromal cells (GBMSCs) cultured in the perfusion bioreactor for 14 days using osteogenic medium. Cell seeded scaffolds cultured in static conditions acted as controls. After 14 days, constructs (42 mm length and 16 mm in diameter) were implanted in critical size defects performed in the tibial bone of six adult goats from which the bone marrow had been collected previously. Explants were retrieved after six and 12 weeks of implantation and characterized using scanning electron microscopy, energy-dispersive spectroscopy, micro-computed tomography and radiographic analysis to assess tissue morphology and calcification. Explants were histologically analyzed, using Hematoxylin & Eosin and Masson Trichrome staining. Results provided relevant information about the performance and functionality of starch with polycaprolactone-goat bone marrow stromal cell constructs in a critical size orthotopic defect performed in a large animal model and demonstrated that culture of the starch with polycaprolactone scaffolds with the autologous cells in perfusion culture provide a good therapy for the healing and regenerative process of bone defects.

Histologic changes in the sinus membrane after maxillary sinus augmentation with simultaneous implant placement using engineered bone graft material

The aim of the study was to analyze the histologic and ultrastructural changes after maxillary sinus augmentation with simultaneous implant placement using engineered bone graft material. In this study, calcium phosphate cement (CPC) scaffolds combined with goat bone marrow stromal cells (BMSCs) were used to fill goat sinus floor space after maxillary sinus floor elevation with simultaneous implant placement comparing with those not filled any grafted materials and used as controls. After a healing period of 3 months, the goat maxillary sinus membrane was examined using light microscopy and scanning electronic microscopy. The results showed that the connective tissue thickness and the epithelium thickness of mucosa were not statistically significant difference between two groups. The tissue engineered bone complex might be an ideal graft for the sinus floor elevation and have no influence on the sinus membrane under the histological and ultrastructural observation.

Transplantation of Goat Bone Marrow Stromal Cells to the Degenerating Intervertebral Discin a Goat Disc Injury Model

Study Design In vivo randomized controlled study in the goat intervertebral disc (IVD) injury model.

In situ functionalization of wet-spun fibre meshes for bone tissue engineering

Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (Si-OH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet-spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet-spun fibre meshes with Si-OH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet-spinning, using a calcium silicate solution as a non-solvent and functionalized in situ with Si-OH groups. In vitro tests, using goat bone marrow stromal cells (GBMSCs), showed that SPCL-Si scaffolds sustained cell viability and proliferation. Furthermore, high ALP activity and matrix production indicated that Si-OH groups improve cellular functionality towards the osteoblastic phenotype. Using this methodology, and assembling several wet-spun fibre meshes, 3D meshes can be developed, aiming at designing osteoconductive/osteoinductive 3D structures capable of stimulating bone ingrowth in vivo.

Differentiation of mesenchymal stem cells in chitosan scaffolds with double micro and macroporosity

Bone Marrow mesenchymal stem cells can be induced to differentiate into osteoblasts to regenerate damaged bone tissue using tissue engineering techniques. In this study, we examine the use of chitosan scaffolds with double pore structure prepared by an innovative method that combines freeze gelation (that produces micropores) and particle leaching out technique (that produces interconnected spherical macropores) seeking to enhance the osteogenic differentiation of goat bone marrow stromal cells (GBMSCs). The double pore architecture of the scaffold was characterized by scanning electron microscopy (SEM), microcomputed tomography and confocal laser scanning microscopy. The obtained hierarchical pore structure allowed very efficient seeding of GBMSCs that are able to occupy the whole volume of the scaffold, showing good adhesion and proliferation. GBMSCs were differentiated into osteoblasts as indicated by alkaline phosphatase activity and osteocalcin expression. The results of this study demonstrate that chitosan scaffold may be promising biomaterial for bone regeneration.

In Vivo Matrix Production by Bone Marrow Stromal Cells Seeded on PLGA Scaffolds for Ligament Tissue Engineering

Ligament tissue engineering based on cell-seeded biomechanically functional constructs is a commonly studied strategy toward native anterior cruciate ligament replacement. Little is known about the survival and differentiation of the seeded cells after the transplantation. We applied retroviral genetic marking to trace implanted cells and studied their differentiation by species-specific immunolabeling of the extracellular matrix produced. Goat bone marrow stromal cells were transduced with a MoMuLV-based vector encoding the DeltaLNGFR gene. Transduced cells were seeded onto poly(lactic-co-glycolic acid) (PLGA) fibers and implanted subcutaneously into nude mice and left for various periods up to 6 weeks. Immunohistochemistry for LNGFR expression showed survival of the seeded cells after transplantation for up to 6 weeks. Immunohistochemistry for collagen type I and III showed the production of fibrous tissue inside the scaffolds. Moreover, using a goat-specific anti-collagen type III, donor-derived matrix could be demonstrated. We conclude that bone marrow stromal cells survived in vivo and at least partially differentiated after implantation.

Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity

We have synthesized methacrylate-endcapped caprolactone networks with tailored water sorption ability, poly(CLMA-co-HEA), in the form of three-dimensional (3D) scaffolds with the same architecture but exhibiting different hydrophilicity character (x(HEA)=0, 0.3, 0.5), and we investigated the interaction of goat bone marrow stromal cells (GBMSCs) with such structures. For this purpose, GBMSCs were seeded and cultured for 3, 7, 14, 21, and 28 days onto the developed scaffolds. Cells have proliferated throughout the whole scaffold volume. Cell adhesion and morphology were analyzed by SEM, whereas cell viability and proliferation was assessed by MTS test and DNA quantification concluding that numbers of cells increased as a function of the culturing time (until day 14) and also with the hydrophobic content in the samples (from 50 to 100% of CLMA). No significant difference between samples with 100% and 70% of CLMA were detected in some cases. Osteoblastic differentiation was followed by assessing the alkaline phosphatase activity of cells, as well as type I collagen and osteocalcin expressions levels until day 21. The three markers were positive at days 14 and 21 when cells were cultured in 100% CLMA substrates which suggests osteoblastic differentiation of mesenchymal stem cells within these scaffolds. On the other hand, when the CLMA content decreases (until 50%), type I collagen and osteocalcin were positive but ALP was negative indicating that the differentiation process is affected by hydrophilic content. We suggest that such system may be useful to extract information on the effect of materials' wettability on the corresponding biological performance in a 3D environment. Such general insights may be relevant in the context of biomaterials selection for tissue engineering strategies.

Chitosan microparticles as injectable scaffolds for tissue engineering

The use of chitosan microparticles as injectable carriers for cell transplantation represents a promising alternative to avoid the drawbacks of the implantation of other forms of three-dimensional (3D) scaffolds seeded with cells. In this study, a 3D construct is obtained in vitro by combining chitosan microparticles crosslinked with genipin and goat bone marrow stromal cells (GBMCs). Cell viability and the morphology of GBMCs were evaluated after culture for 7 and 14 days. Our results show the feasibility of chitosan microparticles as potential injectable scaffolds for tissue engineering and regenerative medicine.

A Study of the Role of Nell-1 Gene Modified Goat Bone Marrow Stromal Cells in Promoting New Bone Formation

Nell-1 is a recently discovered secreted protein with the capacity to promote osteoblastic calvarial cell differentiation and mineralization and induce calvarial bone overgrowth and regeneration in various rodent models. However, the extent of Nell-1 osteoinductivity in large animal cells remains unknown. The objective of the study was to evaluate the feasibility of adenoviral encoding Nell-1 (AdNell-1) gene transfer into primary adult goat bone marrow stromal cells (BMSCs) in vitro and in vivo and to compare the osteoinductive effects with those produced by bone morphogenetic protein-2 (BMP-2), a well established osteoinductive molecule currently utilized for regional gene therapy. AdNell-1-transduced BMSCs expressed Nell-1 protein and underwent osteoblastic differentiation within 2 weeks in vitro, which is comparable to AdBMP-2. After intramuscular injection of nude mice, the AdNell-1- and AdBMP-2-transduced BMSCs revealed new bone formation, while untransduced or AdLacZ-transduced BMSCs showed mainly fibrotic tissue proliferation. At 4 weeks, BMP-2 induced significantly larger bone mass with a mature bone margin and central cavity filled with primarily fatty marrow tissue. Nell-1 samples had significantly less bone mass but were histologically similar to newly formed trabecular bone mixed with chondroid bone-like areas verified by type X collagen (ColX) immunohistochemistry. This distinct difference in histomorphology from the bone mass induced by BMP-2 suggests that there is a potential clinical role/advantage for Nell-1 in skeletal tissue engineering and regeneration.

Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.

Online measurement of oxygen consumption by goat bone marrow stromal cells in a combined cell‐seeding and proliferation perfusion bioreactor

In an effort to produce clinically useful volumes of tissue engineered bone products, a direct perfusion bioreactor system was developed. Perfusion flow rate, flow direction, and the position of the bioreactor are factors that influenced the amounts and homogeneity of the cells seeded on the scaffold surface. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cm(3)) of small-sized macroporous biphasic calcium phosphate (BCP) scaffolds (2-6 mm). Cell load and cell distribution were shown using Methylene Blue block staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate the viability of the cells. Although cells were not distributed homogenously after cell seeding, the scaffolds were covered with a viable, homogeneous cell layer after 25 days of cultivation. The hybrid structures became interconnected, and a dense layer of extracellular matrix formed on and in the scaffolds. Online oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption could possibly be used to estimate GBMSC population doubling times during growth in this bioreactor system. On the basis of our results, we conclude that a direct perfusion bioreactor system is capable of seeding and proliferating GBMSCs on BCP ceramic scaffolds that can be monitored online during cultivation.

The effect of cell-based bone tissue engineering in a goat transverse process model

A disadvantage of traditional posterolateral spinal fusion models is that they are highly inefficient for screening multiple conditions. We developed a multiple-condition model that concentrates on the initial process of bone formation from the transverse process and not on a functional fusion. The effect of bone marrow stromal cells (BMSCs) in four different porous ceramic scaffolds was investigated in this setting. Polyacetal cassettes were designed to fit on the goat transverse process and house four different ceramic blocks, i.e: hydroxyapatite (HA) sintered at 1,150 degrees and 1,250 degrees; biphasic calcium phosphate (BCP) and tricalcium phosphate (TCP). Goat BMSCs (n=10) were cultured and per-operatively seeded autologeously on one of two cassettes implanted per animal. The cassettes were bilaterally mounted on the dorsum of decorticated L2-processes for 9 weeks. To asses the dynamics of bone formation, fluorochrome labels were administered and histomorphometry focused on the distribution of bone in the scaffolds. A clear difference in the extent of bone ingrowth was determined for the different scaffold types. An obvious effect of BMSC seeding was observed in three of four scaffold types, especially in scaffold regions adjacent to the overlying muscle. Generally, the BCP and TCP scaffolds showed better osteoconduction and an increased response to BMSCs administration. In conclusion the model provides a reliable and highly efficient method to study bone formation in cell-based tissue engineering. An effect of cell administration was obvious in three of the four scaffold materials.

Towards Injectable Cell-based Tissue-Engineered Bone: The Effect of Different Calcium Phosphate Microparticles and Pre-Culturing

Bone tissue engineering by combining bone marrow stromal cells (BMSCs) with a porous scaffold is a promising technology. Current major challenges are to upscale the technique for clinical application and to improve the handling characteristics. With respect to minimal invasive surgery, moldable and/or injectable formulations are highly preferable. Ceramic microparticles of different HA/TCP formulations (100/0, 70/30, 60/40, 40/60, and 0/100) with varying surface roughness were sieved to select 200 microg aliquots of the 212-300 microm fraction. Goat BMSCs were seeded on different aliquots one week prior to in vivo implantation. These constructs and remaining cells were cultured for one week. By then, the remaining cells were harvested and resuspended in a specific binder: hyaluronic acid, alginate, or blood plasma, combined with aliquots of 60/40 microparticles peroperatively. All constructs were implanted in nude rats (n = 10) and analyzed for their bone yield histomorphometrically after 6 weeks. All precultured constructs showed consistent bone formation of comparable quantity. No significant differences were observed between the different material compositions. Peroperatively prepared constructs hardly showed any bone formation. The present study demonstrated the osteogenic potential of a tissue- engineered bone substitute made of microparticles of various HA/TCP compositions. There was an obvious advantage when the constructs were pre-cultured.

A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: In vivo bone formation showing proof of concept

In an effort to produce clinically relevant volumes of tissue-engineered bone products, we report a direct perfusion bioreactor system. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cc) of small sized macroporous biphasic calcium phosphate scaffolds (BCP, 2–6 mm). Cell load and cell distribution were shown using methylene blue block staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate viability of the present cells. After 19 days of cultivation, the scaffolds were covered with a viable, homogeneous cell layer. The hybrid structures became interconnected and a dense layer of extracellular matrix was present as visualized by environmental scanning electron microscopy (ESEM). ESEM images showed within the extracellular matrix sphere like structures which were identified as calcium phosphate nodules by energy dispersive X-ray analysis (EDX). On line oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption can be used to estimate GBMSC population doubling times during growth in this bioreactor system. Implantation of hybrid constructs, which were proliferated dynamically, showed bone formation in nude mice after 6 weeks of implantation. On the basis of our results we conclude that a direct perfusion bioreactor system is capable of producing clinically relevant volumes of tissue-engineered bone in a bioreactor system which can be monitored on line during cultivation and show bone formation after implantation in nude mice.

Bone tissue engineering in a critical size defect compared to ectopic implantations in the goat

Since the application of the autologous bone graft, the need for an alternative has been recognized. Tissue engineering (TE) of bone by combining bone marrow stromal cells (BMSCs) with a porous scaffold, is considered a promising technique. In this study we investigated the potential of tissue engineered bone to heal a critical sized defect in the goat. Orthotopic bone formation was compared to ectopic bone formation in comparable constructs. TE constructs were prepared from goat BMSCs and porous biphasic calcium phosphate ceramic scaffolds. These constructs and scaffolds without cells were implanted paired in critical sized iliac wing defects. Comparable samples were implanted intramuscularly. After 9 ( n=7) and 12 ( n=8) weeks implantation, the samples were analyzed histomorphometrically. After 9-weeks implantation in the iliac wing defect, significantly more bone apposition was found in the TE condition. After 12 weeks, the defects were almost completely filled with bone, but no significant advantage of TE was determined anymore. This contrasted with the intramuscular samples where TE implants showed significantly more bone at both time points. In conclusion, bone TE is feasible in critical sized defects. However, when appropriate osteoconductive/inductive materials are applied the effect of cell seeding may be temporary.

Design and fabrication of standardized hydroxyapatite scaffolds with a defined macro-architecture by rapid prototyping for bone-tissue-engineering research.

This investigation describes the production and characterization of calcium phosphate scaffolds with defined and reproducible porous macro-architectures and their preliminary in vitro and in vivo bone-tissue-engineered response. Fugitive wax molds were designed and produced using a rapid prototyping technique. An aqueous hydroxyapatite slurry was cast in these molds. After sintering at 1250 degrees C and then cleaning, dimensional and material characterizations of the scaffolds were performed. The resulting scaffolds represented the design, and their dimensions were remarkably consistent. A texture inherent to the layer-by-layer production of the mold was impressed onto the vertical surfaces of the scaffolds. The surface roughness (R(a)) of the textured surfaces was significantly greater than that of the nontextured surfaces. Material analyses revealed a beta-TCP phase in addition to hydroxyapatite for the molded ceramics. Non-molded control ceramics exhibited only hydroxyapatite. Thirty scaffolds were seeded with culture-expanded goat bone-marrow stromal cells (BMSCs) and implanted subcutaneously in nude mice for 4 or 6 weeks. Histology revealed mineralized bone formation in all the scaffolds for both implantation periods. After 4 weeks, bone was present primarily as a layer on scaffold surfaces. After 6 weeks, the surface bone formation was accompanied by bone budding from the surface and occasional bridging of pores. This budding and bridging bone formation almost always was associated with textured scaffold surfaces. However, the area percentage of bone in pores was similar for the 4- and 6-week implantation periods.