The efficacy of poly-d,l-lactic acid- and hyaluronic acid-coated bone substitutes on implant fixation in sheep.

BACKGROUND/OBJECTIVE: The present study investigated the efficacy of poly-d,l-lactic acid (PDLLA) and hyaluronic acid (HyA) on implant fixation when coated onto hydroxyapatite/beta-tri-calcium phosphate (HA/ßTCP) granules. METHODS: The effect was assessed in a clinically relevant in vivo gap model in sheep. Thus, four titanium implants combined with either allograft (control), pure HA/ßTCP, HyA infiltrated HA/ßTCP, or PDLLA reinforced HA/ßTCP granules were bilaterally inserted into the trabecular bone of the distal femurs in eight sheep. The insertion created a 2-mm peri-implant gap. After 12 weeks, histomorphometry and push-out test was used for quantification of newly formed bone in the gap, bone-implant contact, and implant fixation. RESULTS: The histomorphometric analysis revealed the presence of newly formed bone in all groups, though substitute groups showed fragments of nonabsorbed substitute material. A significant larger bone volume was found in the allograft group versus the HA/ßTCP-PDLLA group (Zone 1), and in Zone 2 a statistically significantly larger bone volume was found in the allograft compared with the HA/ßTCP group. The mechanical properties and the bone-implant contact revealed no statistically significant differences between the groups. CONCLUSION: This study demonstrates that HA/ßTCP granules coated with PDLLA and HyA have similar bone ingrowth and implant fixation as those with allograft, and with mechanical properties resembling those of allograft in advance, they may be considered as alternative substitute materials for bone formation in sheep.

Assessment of activated porous granules on implant fixation and early bone formation in sheep.

BACKGROUND/OBJECTIVE: Despite recent progress in regeneration medicine, the repair of large bone defects due to trauma, inflammation and tumor surgery remains a major clinical challenge. This study was designed to produce large amounts of viable bone graft materials in a novel perfusion bioreactor to promote bone formation. METHODS: Cylindrical defects were created bilaterally in the distal femurs of sheep, and titanium implants were inserted. The concentric gap around the implants was randomly filled either with allograft, granules, granules with bone marrow aspirate (BMA) or bioreactor activated granule (BAG). The viable BAG consisted of autologous bone marrow stromal cells (BMSCs) seeded upon porous scaffold granules incubated in a 3D perfusion bioreactor for 2 weeks prior to surgery. 6 weeks after, the bone formation and early implant fixation were assessed by means of micro-CT, histomorphometry, and mechanical test. RESULTS: Microarchitectural analysis revealed that bone volume fraction and trabecular thickness in the allograft were not statistically different than those (combination of new bone and residue of granule) in the other 3 groups. The structure of the allograft group was typically plate-like, while the other 3 groups were combination of plate and rod. Histomorphometry showed that allograft induced significantly more bone and less fibrous tissue in the concentric gap than the other 3 granule groups, while the bone ingrowth to implant porous surface was not different. No significant differences among the groups were found regarding early implant mechanical fixation. CONCLUSION: In conclusion, despite nice bone formation and implant fixation in all groups, bioreactor activated graft material did not convincingly induce early implant fixation similar to allograft, and neither bioreactor nor by adding BMA credited additional benefit for bone formation in this model.

Efficacy of a small cell-binding peptide coated hydroxyapatite substitute on bone formation and implant fixation in sheep.

Cylindrical critical size defects were created at the distal femoral condyles bilaterally of eight female adult sheep. Titanium implants with 2-mm concentric gaps were inserted and the gaps were filled with one of the four materials: allograft; a synthetic 15-amino acid cell-binding peptide coated hydroxyapatite (ABM/P-15); hydroxyapatite + ßtricalciumphosphate+ Poly-Lactic-Acid (HA/ßTCP-PDLLA); or ABM/P-15+HA/ßTCP-PDLLA. After nine weeks, bone-implant blocks were harvested and sectioned for micro-CT scanning, push-out test, and histomorphometry. Significant bone formation and implant fixation could be observed in all four groups. Interestingly, the microarchitecture of the ABM/P-15 group was significantly different from the control group. Tissue volume fraction and thickness were significantly greater in the ABM/P-15 group than in the allograft group. Bone formation and bone ingrowth to porous titanium implant were not significantly different among the four groups. The ABM/P-15 group had similar shear mechanical properties on implant fixation as the allograft group. Adding HA/ßTCP-PDLLA to ABM/P-15 did not significantly change these parameters. This study revealed that ABM/P-15 had significantly bone formation in concentric gap, and its enhancements on bone formation and implant fixation were at least as good as allograft. It is suggested that ABM/P-15 might be a good alternative biomaterial for bone implant fixation in this well-validated critical-size defect gap model in sheep. Nevertheless, future clinical researches should focus on prospective, randomized, controlled trials in order to fully elucidate whether ABM/P-15 could be a feasible candidate for bone substitute material in orthopedic practices.

The effects of a novel-reinforced bone substitute and Colloss®E on bone defect healing in sheep.

Hydroxyappatite-ß-tricalciumphosphate (HA/ß-TCP) was reinforced with poly(D,L)-lactic acid (PDLLA) to overcome its weak mechanical properties. Two substitutes with porosities of 77% and 81% HA/ß-TCP reinforced with 12 wt % PDLLA were tested in compression. The effects of allograft, substitute (HA/ß-TCP-PDLLA), Colloss®E, and combination of substitute with Colloss®E on bone formation in vivo were evaluated. Cylindrical critical size defects were created at distal femoral condyles bilaterally in sheep. Titanium implant with concentric gap filling with one of the four materials was inserted. After 9 weeks, the sheep were sacrificed. Implants with surrounding bone were harvested and sectioned into two parts: one for microcomputed tomography scanning and push-out test, and one for histomorphometry. The 77% HA/ß-TCP reinforced with PDLLA had similar mechanical properties to human cancellous bone and was significantly stronger than the HA/ß-TCP without PDLLA. Microarchitecture of gap mass was significantly changed after implantation for all groups. Allograft had stronger shear mechanical properties than the other three groups, whereas there were no significant differences between the other three groups. Significant new bone formation could be seen in vivo in all four groups and there were no significant differences between them. The PDLLA-reinforced substitute seems to be good alternative substitute material for bone healing in sheep. Further investigations should be performed to validate this novel substitute material.

Effects of a perfusion bioreactor activated novel bone substitute in spine fusion in sheep.

PURPOSE: To evaluate the effect of a large perfusion-bioreactor cell-activated bone substitute, on a two-level large posterolateral spine fusion sheep model. METHODS: A 50 mm long porous biphasic-calcium-phosphate bone substitute reinforced with poly(D,L-lactide) and, activated with bone marrow derived mononuclear-cells (BMNC) was used. Eighteen sheep were divided into two groups and one group (n = 9) had BMNC-activated bone substitutes and cell-free substitutes implanted. The second group (n = 9) had autograft supplemented with BMNC and regular autograft implanted. The implant material was alternated between spine level L2-L3 and L4-L5 in both groups. MicroCT was used to compare the spine fusion efficacy and bone structure of the two groups as well as the implanted bone substitutes and non-implanted substitutes. RESULTS: After 4½ months six sheep survived in both groups and we found five spine levels were fused when using activated bone substitute compared to three levels with cell-free bone substitute (p = 0.25). Five sheep fused at both levels in the autograft group. A significant increased bone density (p < 0.05) and anisotropy (p < 0.05) was found in the group of activated bone substitutes compared to cell-free bone substitute and no difference existed on the other parameters. The implanted bone substitutes had a significant higher bone density and trabecular thickness than non-implanted bone substitutes, thus indicating that the PLA reinforced BCP had osteoconductive properties (p < 0.05). No effect of the supplemented BMNC to autograft was observed. The autograft group had a significant higher bone density, trabecular thickness and degree of anisotropy than the implanted bone substitutes (p < 0.05), but a lower connectivity density existed (p < 0.05). This indicates that though the activated substitute might have a similar fusion efficacy to autograft, the fusion bridge is not of equal substance. CONCLUSION: We found that bioreactor-generated cell-based bone substitutes seemed superior in fusion ability when compared to cell-free bone substitute and comparable to autograft in fusion ability, but not in bone structure. This combined with the favorable biocompatible abilities and strength comparable to human cancellous bone indicates that it might be a suitable bone substitute in spine fusion procedures.

A 3D in vitro bone organ model using human progenitor cells.

Three-dimensional (3D) organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF) of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0±0.3%), pre/osteoclastic (14.0±1.4%) and mesenchymal/osteoblastic (44.0±8.4%) phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides), resorption (by N-terminus collagen-I telopeptides and phosphate levels) and osteoclastic activity (by TRAP-5b) only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

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.