Sustained release of ancillary amounts of testosterone and alendronate from PLGA coated pericard membranes and implants to improve bone healing.

Testosterone and alendronate have been identified as two bone healing compounds which, when combined, synergistically stimulate bone regeneration. This study describes the development of a novel ultrasonic spray coating for sustained release of ancillary amounts of testosterone and alendronate encapsulated in PLGA 5004A as a carrier. Due to the low amounts of testosterone and alendronate used, sensitive in vitro assays were developed to determine in vitro release. The ultrasonic spray coating technology was optimized for coating titanium screws and pericardial collagen membranes, with the aim to improve osseo-integration and (guided) bone regeneration, respectively, without interfering with their primary mode of action. In vitro release analysis of collagen membranes and screws showed up to 21 days sustained release of the compounds without a burst release. Subsequent preclinical studies in rat and rabbit models indicated that testosterone and alendronate coated membranes and screws significantly improved bone regeneration in vivo. Coated membranes significantly improved the formation of new bone in a critical size calvarial defect model in rats (by 160% compared to controls). Coated screws implanted in rabbit femoral condyles significantly improved bone implant contact (69% vs 54% in controls), bone mineral density (121%) and bone volume (119%) up to 1.3 mm from the implant. Based on the results obtained, we suggest that implants or membranes enabled with local sustained delivery of ancillary amounts of testosterone and alendronate can be a promising system to stimulate local bone regeneration resulting in improved osseo-integration of implants and improved healing of bone defects and fractures.

Osteogenic properties of starch poly(ε-caprolactone) (SPCL) fiber meshes loaded with osteoblast-like cells in a rat critical-sized cranial defect.

Osteoblast-like cells together with a suitable scaffold can aid to the regeneration of bone defects. A suitable scaffold could be starch poly(ε-caprolactone) (SPCL) fiber meshes, which have shown a high potential to support bone formation in previous in vitro and in noncritical sized in vivo studies. The aim of this study was to assess the effect of these scaffolds alone or combined with osteoblast-like cells in the regeneration of a critical-sized cranial defect in male Fisher rats. Empty defects and defects filled with cell-free scaffolds were used as controls groups. Samples were analyzed by microcomputed tomography (micro-CT) and histological analyses. Histological analyses revealed that all study groups showed new bone formation from the defect edges toward the interior of the defects. In addition, bone was formed in the center of the scaffolds, especially in the groups containing preloaded osteoblast-like cells. Micro-CT reconstructions showed that bone formation increased over time and was enhanced with the inclusion of preloaded osteoblast-like cells compared with SPCL scaffolds alone. According to these results, the preloaded osteoblast-like cells contributed to the bone regeneration process in a critical-sized bone defect. Furthermore, SPCL fiber meshes proved to be an osteoconductive material to use for bone regeneration purposes.

Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles.

This study focused on the degradation properties of gelatin microparticles incorporated in calcium phosphate (CaP) cement and the subsequent effect of these composites on bone formation. Positively charged alkaline gelatin (type A) microparticles or negatively charged acidic gelatin (type B) microparticles were incorporated in CaP cement, which was implanted in critical-sized cranial defect in rats and left in place for 2, 4, and 8 weeks. The degradation of the gelatin was monitored using radioiodinated microparticles. After 4 and 8 weeks of implantation, a significantly faster degradation of type A gelatin over type B gelatin was found. Light microscopic analysis of the specimens showed similar bone response concerning implants containing either type A or B gelatin microparticles. At 2 weeks of implantation, a minimal amount of bone formation was observed from the cranial bone toward the implant, while after 8 weeks of implantation an entire layer of newly formed bone was present from the cranial bone toward the implant periphery. Bone ingrowth into the implant was observed at sites of gelatin microparticle degradation, predominantly at the implant periphery. Histomorphometrical evaluation did not reveal significant differences in bone formation between CaP cement incorporated with either type A or B gelatin microparticles during implantation periods up to 8 weeks. In conclusion, this study demonstrates that gelatin type influences the degradation of gelatin microparticles incorporated in CaP cements. However, this difference in degradation and the concomitant subsequent macroporosity did not induce differences in the biological response.

Evaluation of the biocompatibility of calcium phosphate cement/PLGA microparticle composites.

In this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model in rats. Short-term biocompatibility was assessed using pure CaP discs and CaP discs incorporating PLGA microparticles (20% w/w) with and without preincubation in water. Long-term biocompatibility was assessed using CaP discs incorporating varying amounts (5, 10, or 20% w/w) and diameter sizes (small, 0-50 mum; medium, 51-100 mum, or large, 101-200 mum) of PLGA microparticles. The short-term biocompatibility results showed a mild tissue response for all implant formulations, irrespective of disc preincubation, during the early implantation periods up to 12 days. Quantitative histological evaluation revealed that the different implant formulations induced the formation of similar fibrous tissue capsules and interfaces. The results concerning long-term biocompatibility showed that all implants were surrounded by a thin connective tissue capsule (<10 layers of fibroblasts). Additionally, no significant differences in capsule and interface scores were observed between the different implant formulations. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth throughout the implants, indicating PLGA degradation and interconnectivity of the pores. The results demonstrate that CaP/PLGA composites evoke a minimal inflammatory response. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth after 12- and 24-weeks indicating PLGA degradation and interconnectivity of the pores. Therefore, CaP/PLGA composites can be regarded as biocompatible biomaterials with potential for bone tissue engineering and advantageous possibilities of the microparticles regarding material porosity.

Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-beta 1 loaded gelatin microparticles.

This study focused at the potential of transforming growth factor beta 1 (TGF-beta 1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable calcium phosphate (CaP)/gelatin microparticle composites. Therefore, TGF-beta1 loaded composites and non-loaded controls were injected in circular defects as created in the femoral condyles of rabbits and were left in place for 4, 8 and 12 weeks. The specimens were evaluated mechanically (push-out test), and morphologically (scanning electron microscopy (SEM), histology, and histomorphometry). The results showed a gradual increase in mechanical strength with increasing implantation periods. Histological and histomorphometrical evaluation showed similar results for both composite formulations regarding histological aspect, new bone formation and bone/implant contact. However, TGF-beta1 loading of the composites demonstrated a significant effect on composite degradation after twelve weeks of implantation. The results of this study showed that CaP/gelatin composites show excellent osteogenic properties and a rapid increase in mechanical strength. The addition of TGF-beta1 significantly enhances the bone remodeling process.

The cytocompatibility and early osteogenic characteristics of an injectable calcium phosphate cement.

In this study, the cytocompatibility and early osteogenic characteristics of rat bone marrow cells (RBMCs) on injectable calcium phosphate (CaP) cement (Calcibon) were investigated. In addition to unmodified CaP cement discs, 2 other treatments were given to the discs: preincubation in MilliQ and sintering at different temperatures. After primary culture, RBMCs were dropwise seeded on the discs and cultured for 12 days. The samples were evaluated in terms of cell viability, morphology (live and dead assays and scanning electron microscopy (SEM)), cell proliferation (deoxyribonucleic acid (DNA) analyses), early cell differentiation (alkaline phosphatase (ALP) activity), and physicochemical analyses (x-ray diffraction (XRD)). The live and dead, DNA, and SEM results showed that Calcibon discs without any additional treatment were not supporting osteoblast-like cells in vitro. There were fewer cells, and cell layers were detached from the disc surface. Therefore, different preincubation periods and sintering temperatures were evaluated to improve the cytocompatibility of the CaP cement. Preincubating discs in MilliQ for periods of 1, 4, 8, and 12 weeks resulted in the hydrolysis of alpha-tri calcium phosphate (TCP) into an apatite-like structure with some beta-TCP, as shown with XRD, but the material was not cytocompatible. Sintering the discs between 800 degrees C and 1100 degrees C resulted in conversion of alpha-TCP to beta-TCP with some hydroxyapatite and an increase in crystallinity. Eventually, the discs sintered at 1100 degrees C achieved better cell attachment, more-abundant cell proliferation, and earlier differentiation than other sintered (600 degrees C, 800 degrees C, and 1000 degrees C), preincubated, and unmodified specimens. On basis of our results, we conclude that in vivo results with CaP-based cements do not guarantee in vitro applicability. Furthermore, unmodified Calcibon is not cytocompatible in vitro, although preincubation of the material results in a more-favorable cell response, sintering of the material at 1100 degrees C results in the best osteogenic properties. In contrast to in vivo studies, the Calcibon CaP cement is not suitable as a scaffold for cell-based tissue-engineering strategies.

Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles.

In this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20wt% poly (dl-lactic-co-glycolic acid) (PLGA) microparticles were investigated in a rat cranial defect. After 2, 4 and 8 weeks of implantation, implants were evaluated mechanically (push-out test) and morphologically (Scanning Electron Microscopy (SEM) and histology). The results of the push-out test showed that after 2 weeks the shear strength of the implants was 0.44+/-0.44MPa (average+/-sd), which increased to 1.34+/-1.05MPa at 4 weeks and finally resulted in 2.60+/-2.78MPa at 8 weeks. SEM examination showed a fracture plane at the bone-cement interface at 2 weeks, while the 4- and 8-week specimens created a fracture plane into the CaP/PLGA composites, indicating an increased strength of the bone-cement interface. Histological evaluation revealed that the two weeks implantation period resulted in minimal bone ingrowth, while at 4 weeks of implantation the peripheral PLGA microparticles were degraded and replaced by deposition of newly formed bone. Finally, after 8 weeks of implantation the degradation of the PLGA microparticles was almost completed, which was observed by the bone ingrowth throughout the CaP/PLGA composites. On basis of our results, we conclude that the shear strength of the bone-cement interface increased over time due to bone ingrowth into the CaP/PLGA composites. Although the bone-cement contact could be optimized with an injectable CaP cement to enhance bone ingrowth, still the mechanical properties of the composites after 8 weeks of implantation are insufficient for load-bearing purposes.