Regenerating Critical Size Rat Segmental Bone Defects with a Self-Healing Hybrid Nanocomposite Hydrogel: Effect of Bone Condition and BMP-2 Incorporation.

The aim of the current study is to assess the biological performance of self-healing hydrogels based on calcium phosphate (CaP) nanoparticles and bisphosphonate (BP) conjugated hyaluronan (HA) in a critical size segmental femoral bone defect model in rats. Additionally, these hydrogels are loaded with bone morphogenetic protein 2 (BMP-2) and their performance is compared in healthy and osteoporotic bone conditions. Treatment groups comprise internal plate fixation and placement of a PTFE tube containing hydrogel (HABP -CaP) or hydrogel loaded with BMP-2 in two dosages (HABP -CaP-lowBMP2 or HABP -CaP-highBMP2). Twelve weeks after bone defect surgery, bone formation is analyzed by X-ray examination, micro-CT analysis, and histomorphometry. The data show that critical size, segmental femoral bone defects cannot be healed with HABP -CaP gel alone. Loading of the HABP -CaP gel with low dose BMP-2 significantly improve bone formation and resulted in defect bridging in 100% of the defects. Alternatively, high dose BMP-2 loading of the HABP -CaP gel does not improve bone formation within the defect area, but leads to excessive bone formation outside the defect area. Bone defect healing is not affected by osteoporotic bone conditions.

Bioinorganic supplementation of calcium phosphate-based bone substitutes to improve in vivo performance: a systematic review and meta-analysis of animal studies.

Supplementation of CaP-based bone graft substitutes with bioinorganics such as strontium, zinc or silicon is an interesting approach to increase the biological performance in terms of bone regenerative potential of calcium phosphate (CaP)-based bone substitutes. However, the in vivo efficacy of this approach has not been systematically analyzed, yet. Consequently, we performed a systematic review using the available literature regarding the effect of bioinorganic supplementation in CaP-based biomaterials on new bone formation and material degradation in preclinical animal bone defect models and studied this effect quantitatively by performing a meta-analysis. Additional subgroup analyses were used to study the effect of different bioinorganics, animal model, or phase category of CaP-based biomaterial on bone formation or material degradation. Results show that bioinorganic supplementation increases new bone formation (standardized mean difference [SMD]: 1.43 SD, confidence interval [CI]: 1.13-1.73). Additional subgroup analysis showed that strontium, magnesium and silica significantly enhanced bone formation, while zinc did not have any effect. This effect of bioinorganic supplementation on new bone formation was stronger for DCPD or ß-TCP and biphasic CaPs than for HA or α-TCP (p < 0.001). In general, material degradation was slightly hindered by bioinorganic supplementation (mean difference [MD]: 0.84%, CI: 0.01-1.66), with the exception of strontium that significantly enhanced degradation. Overall, bioinorganic supplementation represents an effective approach to enhance the biological performance of CaP-based bone substitutes.

Preclinical evaluation of injectable bone substitute materials.

Injectable bone substitutes (IBSs) represent an attractive class of ready-to-use biomaterials, both ceramic- and polymer-based, as they offer the potential benefit of minimally invasive surgery and optimal defect filling. Although in vitro assessments are the first step in the process of development, the safety and efficacy of an IBS strongly depend on validated preclinical research prior to clinical trials. However, the selection of a suitable preclinical model for performance evaluation of an IBS remains a challenge, as no gold standard exists to define the best animal model. In order to succeed in this attempt, we identified three stages of development, including (a) proof-of-principle, (b) predictive validity and (c) general scientific legitimacy, and the respective criteria that should be applied for such selection. The second part of this review provides an overview of commonly used animals for IBSs. Specifically, scientific papers published between January 1996 and March 2012 were retrieved that report the use of preclinical models for the evaluation of IBSs in situations requiring bone healing and bone augmentation. This review is meant not only to describe the currently available preclinical models for IBS application, but also to address critical considerations of such multi-factorial evaluation models (including animal species, strain, age, anatomical site, defect size and type of bone), which can be indicative but in most cases edge away from the human reality. Consequently, the ultimate goal is to guide researchers toward a more careful and meaningful interpretation of the results of experiments using animal models and their clinical applications.

Osteogenic capacity of human BM-MSCs, AT-MSCs and their co-cultures using HUVECs in FBS and PL supplemented media.

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) and human adipose tissue-derived mesenchymal stem cells (AT-MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large-scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS- and PL-supplemented media on osteogenesis in stem cell mono- and co-cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL- compared to FBS-supplemented media in mono- and co-cultures for both BM-MSCs and AT-MSCs. In addition, calcium deposition was cell type dependent and decreased for BM-MSCs but increased for AT-MSCs in PL-supplemented medium in both mono- and co-cultures. Based on the effects of co-cultures, BM-MSCs/HUVECs enhanced osteogenesis compared to BM-MSCs monocultures in both FBS- and PL-supplemented media whereas AT-MSCs/HUVECs showed similar results compared to AT-MSCs monocultures.

Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol.

The murine-derived MC3T3-E1 cell line provided by the American Type Culture Collection (ATCC) is a well-known osteogenic cell culture model system to test materials in vitro. However, the effect of passaging on its mineralization capacity has never been described and their culture supplements can be further optimized. Therefore, we evaluated the influence of the passage number and different osteogenic culture supplements, including ascorbic acid (AsAP) and dexamethasone (Dex) on the osteogenic capacity of MC3T3-E1 cells. This capacity was measured by the deposited calcium, the alkaline phosphatase activity, and the expression of osteogenic-related genes, including bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The results indicated that the mineralization capacity of MC3T3-E1 cells significantly decreased during passaging and got exhausted at passage 34, as assessed by measuring calcium deposition after 28 days of osteogenic induction. Moreover, the combination of AsAP and Dex triggered significantly more mineralization in MC3T3-E1 cells than the ATCC recommended addition of AsAP alone, as indicated by increased calcium deposition and higher expression of BSP and OPN. However, Dex alone could not trigger this effect, but only in combination with the AsAP, which indicates that Dex has no direct effect on mineralization. In conclusion, the passage number of MC3T3-E1 cells is of great importance and the use of cells above 30 passages should be avoided. In addition, the favored osteogenic supplements providing an improved osteogenic differentiation of MC3T3-E1 cells are the combination of AsAP and Dex.

Resolution, sensitivity, and in vivo application of high-resolution computed tomography for titanium-coated polymethyl methacrylate (PMMA) dental implants.

OBJECTIVES: The aims of this study were (i) to determine the spatial resolution and sensitivity of micro- versus nano-computed tomography (CT) techniques and (ii) to validate micro- versus nano-CT in a dog dental implant model, comparative to histological analysis. MATERIAL AND METHODS: To determine spatial resolution and sensitivity, standardized reference samples containing standardized nano- and microspheres were prepared in polymer and ceramic matrices. Thereafter, 10 titanium-coated polymer dental implants (3.2 mm in Ø by 4 mm in length) were placed in the mandible of Beagle dogs. Both micro- and nano-CT, as well as histological analyses, were performed. RESULTS: The reference samples confirmed the high resolution of the nano-CT system, which was capable of revealing sub-micron structures embedded in radiodense matrices. The dog implantation study and subsequent statistical analysis showed equal values for bone area and bone-implant contact measurements between micro-CT and histology. However, because of the limited sample size and field of view, nano-CT was not rendering reliable data representative of the entire bone-implant specimen. CONCLUSIONS: Micro-CT analysis is an efficient tool to quantitate bone healing parameters at the bone-implant interface, especially when using titanium-coated PMMA implants. Nano-CT is not suitable for such quantification, but reveals complementary morphological information rivaling histology, yet with the advantage of a 3D visualization.

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Cryo DualBeam Focused Ion Beam-Scanning Electron Microscopy to Evaluate the Interface Between Cells and Nanopatterned Scaffolds.

With the advance of nanotechnology in biomaterials science and tissue engineering, it is essential that new techniques become available to observe processes that take place at the direct interface between tissue and scaffold materials. Here, Cryo DualBeam focused ion beam-scanning electron microscopy (FIB-SEM) was used as a novel approach to observe the interactions between frozen hydrated cells and nanometric structures in high detail. Through a comparison of images acquired with transmission electron microscopy (TEM), conventional FIB-SEM operated at ambient temperature, and Cryo DualBeam FIB-SEM, the advantages and disadvantages of each technique were evaluated. Ultrastructural details of both (extra)cellular components and cell organelles were best observe with TEM. However, processing artifacts such as shrinkage of cells at the substrate interface were introduced in both TEM and conventional FIB-SEM. In addition, the cellular contrast in conventional FIB-SEM was low; consequently, cells were difficult to distinguish from the adjoining substrate. Cryo DualBeam FIB-SEM did preserve (extra)cellular details like the contour, cell membrane, and mineralized matrix. The three described techniques have proven to be complementary for the evaluation of processes that take place at the interface between tissue and substrate.

The influence of surface roughness on the displacement of osteogenic bone particles during placement of titanium screw-type implants.

BACKGROUND: Previously, we demonstrated that bone debris, which is translocated during dental implant placement, has osteogenic potential. Therefore, it was hypothesized that implant surface roughness can influence the amount of translocated bone debris/particles and thereby the osteogenic response. MATERIAL AND METHODS: Small titanium implants were left turned (smooth) or blasted and acid etched. The implants were placed in fresh cadaver bone. After explantation, the implants were incubated in a culture medium containing ß-glycerophosphate and dexamethasone up to 24 days. Subsequently, histology, scanning electron microscopy (SEM), DNA analysis, and calcium (Ca) content measurements were performed. RESULTS: For both types of implant during implant placement, bone particles were translocated because of inherent roughness of the implant. SEM and histology confirmed the presence of a bone-like tissue on the surface of both types of implants, as also confirmed by DNA and Ca measurements. However, the significantly higher roughness of the etched implants accounted for more bone debris and accordingly elevated osteogenic response. Control samples, which had not been placed into bone, did not show mineralization in the same medium. CONCLUSION: The present study, for the first time, demonstrated that implant surface roughness can increase the amount of the translocated bone particles and thereby also have a beneficial effect on the osteogenic response of these bone particles. It is hypothesized that these bone fragments behave like miniature auto-grafts and thereby play a significant role to enhance peri-implant osteogenesis. Optimization of surface topography should be evaluated to take advantage of this additional effect of surface roughness.

The effect of platelet-rich plasma in vitro on primary cells: rat osteoblast-like cells and human endothelial cells.

The aim of this study was to evaluate the effects of standardized platelet-rich plasma (PRP) concentrates from 10 human donors on cellular behavior. The standardized PRPs used were fivefold average and fivefold maximum baseline values in whole blood. Both these standardized PRPs were characterized by determining platelet numbers and subsequently growth factor concentrations in activated PRPs, called PRP derivatives. Platelet numbers in both types of standardized PRPs were significantly increased compared with whole blood. Further, both PRP derivatives contained significantly higher concentrations of platelet-derived growth factor-AA, platelet-derived growth factor-AB, and transforming growth factor-beta 1. Vascular endothelial growth factor concentrations were significantly elevated in only the most concentrated PRP derivative. Cell culture experiments with osteoblast-like cells showed that both PRP derivatives stimulated cell proliferation without inducing cell differentiation, whereas tube formation in endothelial cell cultures was significantly increased by adding low volume percentages of PRP derivative (2%–8%). Consequently, it can be concluded that there is no direct relationship between the number of platelets and the level of growth factors released from these platelets. PRP derivatives have the potency to stimulate angiogenesis dose dependently, while lacking the capacity to induce osteogenic differentiation. Yet, the proliferation of osteoblast-like cells can significantly be enhanced by supplementation of PRP derivatives.

In vivo bone biocompatibility and degradation of porous fumarate-based polymer/alumoxane nanocomposites for bone tissue engineering.

The objective of this study was to determine how the incorporation of surface-modified alumoxane nanoparticles into a biodegradable fumarate-based polymer affects in vivo bone biocompatibility (characterized by direct bone contact and bone ingrowth) and in vivo degradability. Porous scaffolds were fabricated from four materials: poly(propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA) polymer alone; a macrocomposite consisting of PPF/PF-DA polymer with boehmite microparticles; a nanocomposite composed of PPF/PF-DA polymer and mechanically reinforcing surface-modified alumoxane nanoparticles; and a low-molecular weight PPF polymer alone (tested as a degradation control). Scaffolds were implanted in the lateral femoral condyle of adult goats for 12 weeks and evaluated by micro-computed tomography and histological analysis. For all material groups, small amounts of bone, some soft tissue, and a few inflammatory elements were observed within the pores of scaffolds, though many pores remained empty or filled with fluid only. Direct contact between scaffolds and surrounding bone tissue was also observed in all scaffold types, though less commonly. Minimal in vivo degradation occurred during the 12 weeks of implantation in all materials except the degradation control. These results demonstrate that the incorporation of alumoxane nanoparticles into porous PPF/PF-DA scaffolds does not significantly alter in vivo bone biocompatibility or degradation.

In vitro and in vivo evaluation on the bioactivity of ZnO containing nano-hydroxyapatite/chitosan cement.

A ZnO containing nano-hydroxyapatite/chitosan (n-HA/CS) cement was developed and its bone formation ability was investigated in vitro and in vivo. The physico-chemical properties of the cement were determined in terms of pH variation during and after setting, injectability and wettability. The results indicated that, the pH varied from 7.04 to 7.12 throughout the soaking of the cement in distilled water. The injectability was excellent during the first 4 min, but the cement became less injectable or even not injectable at all after 7 min setting. The static contact angle of the cement against water was 53.5 +/- 2.7 degrees . The results of immersion tests in simulated body fluid (SBF) indicated that the cement exhibited excellent bone-like apatite forming ability. In vivo studies, involving the installation of the cement of tibial-bone defects in rabbit tibia revealed an inflammatory response around the cement at 3 days of implantation. After 4 weeks, the inflammation began to disappear and the cement had bound to the surrounding host bone. Radiological examination also confirmed that the ZnO containing n-HA/CS cement significantly induced new bone formation. These results suggest that the ZnO containing n-HA/CS cement may be beneficial to enhance bone regeneration in osseous defect sites.

Hard tissue formation of STRO-1-selected rat dental pulp stem cells in vivo.

The objective of this study was to examine hard tissue formation of STRO-1-selected rat dental pulp-derived stem cells, seeded into a calcium phosphate ceramic scaffold, and implanted subcutaneously in mice. Previously, STRO-1 selection was used to obtain a mesenchymal stem cell progenitor subpopulation from primary dental pulp-derived stem cells. In the current study, these cells were cultured with three different media: "BMP-plus" medium containing dexamethasone and 100 ng/mL of rhBMP-2, "odontogenic" medium containing dexamethasone, and "control" medium without supplements. The cell-scaffold complexes were cultured in these media for 1, 4, or 8 days before implantation. Histological analysis demonstrated that the cultures with BMP-plus and 4 days of culture gave the highest percentage of hard tissue formation per implant (36 +/- 9% of pore area). Real-time PCR confirmed these results. In conclusion, STRO-1-selected dental pulp stem cells show effective hard tissue formation in vivo, and a short in vitro culture period and addition of BMP-2 can enhance this effect.

Fabrication and in vitro degradation of porous fumarate-based polymer/alumoxane nanocomposite scaffolds for bone tissue engineering.

In this work, the fabrication and in vitro degradation of porous fumarate-based/alumoxane nanocomposites were evaluated for their potential as bone tissue engineering scaffolds. The biodegradable polymer poly (propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA), a macrocomposite composed of PPF/PF-DA and boehmite microparticles, and a nanocomposite composed of PPF/PF-DA and surface-modified alumoxane nanoparticles were used to fabricate porous scaffolds by photo-crosslinking and salt-leaching. Scaffolds then underwent 12 weeks of in vitro degradation in phosphate buffered saline at 37 degrees C. The presence of boehmite microparticles and alumoxane nanoparticles in the polymer inhibited scaffold shrinkage during crosslinking. Furthermore, the incorporation of alumoxane nanoparticles into the polymer limited salt-leaching, perhaps due to tighter crosslinking within the nanocomposite. Analysis of crosslinking revealed that the acrylate and overall double bond conversions in the nanocomposite were higher than in the PPF/PF-DA polymer alone, though these differences were not significant. During 12 weeks of in vitro degradation, the nanocomposite lost 5.3% +/- 2.4% of its mass but maintained its compressive mechanical properties and porous architecture. The addition of alumoxane nanoparticles into the fumarate-based polymer did not significantly affect the degradation of the nanocomposite compared with the other materials in terms of mass loss, compressive properties, and porous structure. These results demonstrate the feasibility of fabricating degradable nanocomposite scaffolds for bone tissue engineering by photo-crosslinking and salt-leaching mixtures of fumarate-based polymers, alumoxane nanoparticles, and salt microparticles.

The kinetic and biological activity of different loaded rhBMP-2 calcium phosphate cement implants in rats.

The healing of large bone defects can be improved by osteogenic bone graft substitutes, due to growth factor inclusion. A sustained release of these growth factors provides more efficient bioactivity when compared with burst release and might reduce the dose required for bone regeneration, which is desirable for socioeconomical and safety reasons. In this study, we compared different rhBMP-2 loadings in a sustained release system of CaP cement and PLGA-microparticles and were able to couple kinetic to biological activity data. Fifty-two rats received a critical-size cranial defect, which was left open or filled with the cement composites. The implants consisted of plain, high, and five-fold lower dose rhBMP-2 groups. Implantation time was 4 and 12 weeks. Longitudinal in vivo release was monitored by scintigraphic imaging of (131)I-labeled rhBMP-2. Quantitative analysis of the scintigraphic images revealed a sustained release of (131)I-rhBMP-2 for both doses, with different release profiles between the two loadings. However, around 70% of the initial dose was retained in both implant formulations. Although low amounts of rhBMP-2 were released (2.4 +/- 0.8 mug in 5 weeks), histology showed defect bridging in the high-dose implants. Release out of the low-dose implants was not sufficient to enhance bone formation. Implant degradation was limited in all formulations, but was mainly seen in the high-dose group. Low amounts of sustained released rhBMP-2 were sufficient to bridge critically sized defects. A substantial amount of rhBMP-2 was retained in the implants because of the slow release rate and the limited degradation.

Effect of platelet-rich plasma on the early bone formation around Ca-P-coated and non-coated oral implants in cortical bone.

OBJECTIVES: The purpose of the present study was to investigate the effect of local application of platelet-rich plasma (PRP) on the early healing of cortical bone around Ti implants with two different surface configurations. MATERIAL AND METHODS: Six goats were used in this study. PRP fractions were obtained from a venous blood sample of the goats and administered immediately before implant insertion. PRP was applied via gel preparation and installation of the gel into the implant site, or via dipping of the implants in PRP fraction before insertion. A total of 36 implants (18 non-coated and 18 Ca-P-coated) were placed into the tibial cortical bone. The animals were sacrificed at 6 weeks after implantation and implants with surrounding tissue were prepared for histological examination. Histomorphometrical variables like the percentage of implant surface with direct bone-implant contact and the percentage of new and old bone adjacent to the implant were evaluated. RESULTS: More interfacial bone-to-implant contact was observed for all the three groups of Ca-P-coated implants and the Ti/PRP liquid group. All groups revealed similar percentages of old and new bone adjacent to the implant. CONCLUSIONS: It was concluded that the additional use of PRP did not have any effect on the early cortical bone response to the Ca-P-coated implants, while PRP in a liquid form showed a tendency to increase bone apposition to roughened titanium implants.

STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.

In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation.

This study instituted a unique approach to bone tissue engineering by combining effects of mechanical stimulation in the form of fluid shear stresses and the presence of bone-like extracellular matrix (ECM) on osteodifferentiation. Rat marrow stromal cells (MSCs) harvested from bone marrow were cultured on titanium (Ti) fiber mesh discs for 12 days in a flow perfusion system to generate constructs containing bone-like ECM. To observe osteodifferentiation and bone-like matrix deposition, these decellularized constructs and plain Ti fiber meshes were seeded with MSCs (Ti/ECM and Ti, respectively) and cultured in the presence of fluid shear stresses either with or without the osteogenic culture supplement dexamethasone. The calcium content, alkaline phosphatase activity, and osteopontin secretion were monitored as indicators of MSC differentiation. Ti/ECM constructs demonstrated a 75-fold increase in calcium content compared with their Ti counterparts after 16 days of culture. After 16 days, the presence of dexamethasone enhanced the effects of fluid shear stress and the bone-like ECM by increasing mineralization 50-fold for Ti/ECM constructs; even in the absence of dexamethasone, the Ti/ECM constructs exhibited approximately a 40-fold increase in mineralization compared with their Ti counterparts. Additionally, denatured Ti/ECM* constructs demonstrated a 60-fold decrease in calcium content compared with Ti/ECM constructs after 4 days of culture. These results indicate that the inherent osteoinductive potential of bone-like ECM along with fluid shear stresses synergistically enhance the osteodifferentiation of MSCs with profound implications on bone-tissue-engineering applications.

Reconstruction of the mandible with a poly(D,L-lactide) scaffold, autogenous corticocancellous bone graft, and autogenous platelet-rich plasma: an animal experiment.

An animal study is presented, evaluating a method of mandibular reconstruction using a poly(D,Llactide) (PDLLA) scaffold. Six goats underwent a continuity resection of the mandibular angle. The defect was bridged with a preshaped PDLLA scaffold, filled with an autogenous particulate bone graft from the anterior iliac crest, and fixed with two preshaped titanium plates. To accelerate bone healing, autogenous platelet-rich plasma was mixed with the particulate bone graft. All goats had an uneventful healing. The osteosynthesis system withstood immediate loading for a period of 6 weeks until sacrifice. The particulate bone grafts within the PDLLA scaffold, which appeared to be narrowed, showed considerable resorption and replacement by fibrous tissue. In all goats, however, callus formation along the reconstructed segment was seen, providing bony continuity and maintaining the original contour of the reconstructed segment. Thus, the technique used may provide an alternative for reconstruction with revascularized composite flaps with less associated donor site morbidity.

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone.

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.