Efficacy of bioreactor-activated bone substitute with bone marrow nuclear cells on fusion rate and fusion mass microarchitecture in sheep.

Bioreactors have been used for bone graft engineering in pre-clinical investigations over the past 15 years. The ability of bioreactor-incubated bone marrow nuclear cells (BMNCs) to enhance bone-forming potential varies significantly, and the three-dimensional (3D) distribution of BMNCs within the scaffold is largely unknown. The aims of this study were (1) to investigate the efficacy of a carbonated hydroxyapatite (CHA) with/without BMNCs on spine fusion rate and fusion mass microarchitecture using a highly challenging two-level posterolateral spine fusion without instrumentation; and (2) to evaluate 3D distribution of BMNCs within scaffolds characterized by immunohistochemistry. Fusion rate and fusion mass were quantified by micro-CT, microarchitectural analysis, and histology. While the homogenous 3D distribution of BMNCs was not observed, BMNCs were found to migrate towards a substitute core. In the autograft group, the healing rate was 83.3%, irrespective of the presence of BMNCs. In the CHA group, also 83.3% was fused in the presence of BMNCs, and 66.7% fused without BMNCs. A significant decrease in the fusion mass porosity (p = .001) of the CHA group suggested the deposition of mineralized bone. The autograft group revealed more bone, thicker trabeculae, and better trabecular orientation but less connection compared to the CHA group. Immunohistochemistry confirmed the ability of bioreactors to incubate a large-sized substitute coated with viable BMNCs with the potential for proliferation and differentiation. These findings suggested that a bioreactor-activated substitute is comparable to autograft on spine fusion and that new functional bone regeneration could be achieved by a combination of BMNCs, biomaterials, and bioreactors.

3D perfusion bioreactor-activated porous granules on implant fixation and early bone formation in sheep.

Early fixation of total joint arthroplasties is crucial for ensuring implant survival. An alternative bone graft material in revision surgery is needed to replace the current gold standard, allograft, seeing that the latter is associated with several disadvantages. The incubation of such a construct in a perfusion bioreactor has been shown to produce viable bone graft materials. This study aimed at producing larger amounts of viable bone graft material (hydroxyapatite 70% and ß-tricalcium-phosphate 30%) in a novel perfusion bioreactor. The abilities of the bioreactor-activated graft material to induce early implant fixation were tested in a bilateral implant defect model in sheep, with allograft as the control group. Defects were bilaterally created in the distal femurs of the animals, and titanium implants were inserted. The concentric gaps around the implants were randomly filled with either allograft, granules, granules with bone marrow aspirate or bioreactor-activated graft material. Following an observation time of 6 weeks, early implant fixation and bone formation were assessed by micro-CT scanning, mechanical testing, and histomorphometry. Bone formations were seen in all groups, while no significant differences between groups were found regarding early implant fixation. The microarchitecture of the bone formed by the synthetic graft materials resembled that of allograft. Histomorphometry revealed that allograft induced significantly more bone and less fibrous tissue (p < 0.05). In conclusion, bone formation was observed in all groups, while the bioreactor-activated graft material did not reveal additional effects on early implant fixation comparable to allograft in this model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2465-2476, 2017.

The efficacy of custom-made porous hydroxyapatite prostheses for cranioplasty: evaluation of postmarketing data on 2697 patients.

PURPOSE: Cranioplasty is a surgical intervention aimed at reestablishing the integrity of skull defects. Autologous bone is still considered the treatment of choice for cranioplasty. The aims of this study were to characterize and evaluate the efficacy of porous hydroxyapatite (HA) to fill skull defects based on its biomimetic characteristics. METHODS: The authors analyzed the postmarketing data of all patients treated with custom-made porous HA prostheses (CustomBone Service). Characterization data in terms of physicochemical analysis and mechanical performance of the porous HA prostheses were also reported. RESULTS: The low incidence of adverse events (5.72%) due to the use of HA porous custom-made prostheses for cranioplasty is related to the biomimetic performance of the prostheses. The composition and morphology of the porosity enable it to be a useful biomimetic prosthesis for the reconstruction of large and complex skull defects, also able to promote osteointegration. CONCLUSIONS: These collected and analyzed data demonstrate that porous HA is a suitable material to produce custom-made prostheses to repair craniolacunia. It is a biomimetic implant well-tolerated in both adult and pediatric patients and has been shown to be an effective and good alternative for cranial reconstruction.

Engineering of large osteogenic grafts with rapid engraftment capacity using mesenchymal and endothelial progenitors from human adipose tissue.

We investigated whether the maintenance in culture of endothelial and mesenchymal progenitors from the stromal vascular fraction (SVF) of human adipose tissue supports the formation of vascular structures in vitro and thereby improves the efficiency and uniformity of bone tissue formation in vivo within critically sized scaffolds. Freshly-isolated human SVF cells were seeded and cultured into hydroxyapatite scaffolds (1 cm-diameter, 1 cm-thickness) using a perfusion-based bioreactor system, which resulted in maintenance of CD34(+)/CD31(+) endothelial lineage cells. Monolayer-expanded isogenic adipose stromal cells (ASC) and age-matched bone marrow stromal cells (BMSC), both lacking vasculogenic cells, were used as controls. After 5 days in vitro, SVF-derived endothelial and mesenchymal progenitors formed capillary networks, which anastomosed with the host vasculature already 1 week after ectopic nude rat implantation. As compared to BMSC and ASC, SVF-derived cells promoted faster tissue ingrowth, more abundant and uniform bone tissue formation, with ossicles reaching a 3.5 mm depth from the scaffold periphery after 8 weeks. Our findings demonstrate that maintenance of endothelial/mesenchymal SVF cell fractions is crucial to generate osteogenic constructs with enhanced engraftment capacity. The single, easily accessible cell source and streamlined, bioreactor-based process makes the approach attractive towards manufacturing of clinically relevant sized bone substitute grafts.

Structural characterization and mechanical performance of calcium phosphate scaffolds and natural bones: a comparative study.

PURPOSE: The knowledge of the mechanical response of bones and their substitutes is pertinent to numerous medical problems. Understanding the effects of mechanical influence on the body is the first step toward developing innovative treatment and rehabilitation concepts for orthopedic disorders. METHODS: This was a comparative study of 5 synthetic scaffolds based on porous calcium phosphates and natural bones, with regard to their microstructural, chemical, and mechanical characterizations. The structural and chemical characterizations of the scaffolds were examined by means of X-ray diffraction, scanning electron microscopy, and X-ray spectroscopy analysis. The mechanical characterization of bones and bone graft biomaterials was carried out through compression tests using samples with noncomplex geometry. RESULTS: Analysis of the chemical composition, surface features, porosity, and compressive strength indicates that hydroxyapatite-based materials and trabecular bone have similar properties.

Comparison of two carbonated apatite ceramics in vivo.

Carbonated apatite ceramics, with a composition similar to that of bone mineral, are potentially interesting synthetic bone graft substitutes. In the present study, two porous carbonated apatite ceramics were developed, characterized and tested for their bone repair capacity and osteoinductive potential in a goat model. Although the two ceramics were prepared from a similar starting powder, their physico-chemical and structural characteristics differed as a consequence of different preparation methods. Both ceramics had an open and interconnected porous structure with a porosity of about 80%. The morphology of the surface of CA-A and CA-B at the submicron level differed significantly: CA-A consisted of irregular grains with a size of 5-20microm, with 1-10microm large micropores among the grains, whereas CA-B surface consisted of much smaller and regular shaped grains (0.05-0.5microm), with most micropores smaller than 1microm. The specific surface area of CA-B was about 10 times larger than that of CA-A due to its significantly smaller grain size. CA-A and CA-B ceramics contained 3 and 5 wt.% of B-type carbonated apatite, respectively. Although neither ceramic succeeded in completely bridging the 17mm iliac wing defect with new bone after 12weeks of implantation, CA-A showed significantly more bone formation in the pores of the implant than CA-B. The total area percentage of new bone in the total defect area was 12.7+/-1.81 and 5.51+/-1.37 (mean+/-SEM) for CA-A and CA-B, respectively. Intramuscular implantation of the ceramics led to ectopic bone formation by CA-A in all three implanted specimens, in contrast to CA-B, where no new bone was observed in any of the 11 animals. CA-A showed a more pronounced degradation than CA-B both in vitro and in vivo at both implantation sites, which was unexpected based on the physico-chemical and structural properties of the two ceramics. Both physico-chemical and structural properties of the ceramics could, dependently or independently, have affected their in vivo behaviour, emphasizing the importance to control individual parameters for successful bone repair.

Comparative in vivo study of six hydroxyapatite-based bone graft substitutes.

Improvement of synthetic bone graft substitutes as suitable alternatives to a patient's own bone graft remains a challenge in biomaterials research. Our goal was to answer the question of whether improved osteoinductivity of a material would also translate to better bone-healing orthotopically. Three porous biphasic calcium phosphate (BCP) ceramics (BCPA, BCPB, and BCPC), consisting of hydroxyapatite and beta-tricalcium phosphate, a porous biphasic calcium phosphate ceramic reinforced with a bioresorbable polylactic acid to improve its mechanical properties (BCPC+), a pure hydroxyapatite ceramic (HA), and a carbonated apatite ceramic (CA) were implanted intramuscularly and orthotopically by using a transverse process model in 11 goats for 12 weeks. BCPA and BCPB had similar chemical composition but differed in their microstructure. BCPB was not osteoinductive at all, but BCPA induced ectopic bone formation in 9 of 11 animals. Orthotopically, BCPA performed better than BCPB in both the amount and rate of bone formation. BCPC, similar to BCPA structurally and physicochemically, showed comparable results ectopically and orthotopically. Addition of resorbable polymer to BCPC made the material less osteoinductive (4 of 11 animals) and delayed bone formation orthotopically. Neither HA nor CA were osteoinductive, and their orthotopic performance was inferior to the osteoinductive ceramics. The results of the present study showed that material-derived osteoinduction significantly enhanced bone healing orthotopically, and that this material property appeared more sensitive for predicting orthotopic performance than physicochemical and structural characteristics.

SEM and 3D synchrotron radiation micro-tomography in the study of bioceramic scaffolds for tissue-engineering applications.

Different biomaterials have been proposed as scaffolds for the delivery of cells and/or biological molecules to repair or regenerate damaged or diseased bone tissues. Particular attention is being given to porous bioceramics that mimic trabecular bone chemistry and structure. Chemical composition, density, pore shape, pore size, and pore interconnection are elements that have to be considered to improve the efficiency of these biomaterials. Commonly, two-dimensional (2D) systems of analysis such as scanning electron microscope (SEM) are used for the characterization and comparison of the scaffolds. Unfortunately, these systems do not allow a complete investigation of the three-dimensional (3D) spatial structure of the scaffold. In this study, we have considered two different techniques, that is, SEM and 3D synchrotron radiation (SR) micro-CT to extract information on the geometry of two hydroxyapatite (HA) bioceramics with identical chemical composition but different micro-porosity, pore size distribution, and pore interconnection pathway. The two scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold FB), and (b) foaming (scaffold EP). Both scaffolds showed structures suitable for tissue-engineering applications, but scaffold EP appeared superior with regard to interconnection of pores, surface on which the new bone could be deposited, and percentage of volume available to bone deposition.

Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics.

Purpose of this study was the analysis of the role of density and pore interconnection pathway in scaffolds to be used as bone substitutes. We have considered 2 hydroxyapatite bioceramics with identical microstructure and different macro-porosity, pore size distribution and pore interconnection pathway. The scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold A), and (b) foaming (scaffold B). Bone ingrowth within the two bioceramics was obtained using an established model of in vivo bone formation by exogenously added osteoprogenitor cells. The histological analysis of specimens at different time after in vivo implantation revealed in both materials a significant extent of bone matrix deposition. Interestingly enough, scaffold B allowed a faster occurrence of bone tissue, reaching a steady state as soon as 4 weeks. Scaffold A on the other hand reached a comparable level of bone formation only after 8 weeks of in vivo implantation. Both scaffolds were well vascularised, but larger blood vessels were observed in scaffold A. Here we show that porosity and pore interconnection of osteoconductive scaffolds can influence the overall amount of bone deposition, the pattern of blood vessels invasion and finally the kinetics of the bone neoformation process.