Preparation Of A Novel Macroporous Self-setting Bone Substitute And Its Effect On Healing Of A Bone Defect In Osteoporotic Goats

BackgroundOsteoporosis is now becoming much more prevelent than ever, especially in our country, and the number of the people who suffers osteoporosis is exceeding 90 million, therefore, doctors are facing more patients with osteoporotic bone defects. Under circumstance of osteoporosis, osteoclasts are more active, and the ability of bone resorption is stronger than that of bone formation. So healing of this kind of bone defect is impaired, and cure of the bone defect is more diffucult. A bone-grafting procedure is a successful care to treat the bone defects, but there are many limitations with the materials used in the operation. Although improved effect on bone defects has been obtained by using carriers with growth factors, properties of the carriers still need to be upgraded. Normal bone defects can heal fast with rhBMP-2/CPC, but limit factor-release ability of CPC is not enough for osteoporotic bone defects because high concentrations of the growth factors are needed for osteoporotic bone defects. When degradable microspheres loaded with growth factors are mixed wih CPC to construct a macroporous composite, not only the release ability of the composite can be enhanced, but also the degradation rate of it is speeded up. But PLGA that is the most used material for microspheres has some limitations: 1. Slow degradatrion rate hampers new bone ingrowth. 2. Degradation products are harmful to cells and tissues around the materials. GMs, which are widely used as drug carriers, are biocompatible and controlled-degradable after crosslinking, but cytotoxic of the crosslinkers (such as GA) used for parepairing GMs is still an obstacle. The problem will be solved by a new bio-crosslinker—genipin, therefore, GMs may replace PLGA microspheres as a good additive to improve the properties of CPC.Objective1. To prepare GMs by using GP as a crosslinker and to evaluate the characteristics of the GMs for construction of macroporous CPC.2. To investigate the best proportion of GMs and CPC powers in the composite macroporous CPC by analyzing porosity, biomechabical property and the result of XRD. To evaluate the biocompatibility of the composite in vitro and in vivo.3. To study the controlled-release characteristic of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC. To evaluate the bone-induction ability of the both composite in vitro and in vivo.4. To evaluate the effects of the new artificial bone on bone mineral density, biomechanical property, micro-architecture of osteoporotic goat vertebral and healing of ostoporotic bone defect in vivo.Materials and methods1. GMs, prepared by the improved emulsified cold-condensation method, were crosslinked by GP and GA, respectively. After being dispersed in PBS, two kinds of microspheres with the 60% degree of crosslinking were compared in terms of morphology, swelling and degrading properties;BMP-2 were loaded into the GP and GA microspheres, and encapsulation rate,drug loading and releasing capacity were measured; 100%,50% and 25% leaching liquid of GP and GA microspheres were cultured with rat osteoblast(DMEM group as the control), respectively, and cell proliferation were measured by MTT method to grade the cell cytotoxicity.2. Macroporous CPC was developed using genipin-crosslinked GMs with three weight ratios (0 wt%, 2.5 wt% and 5 wt%). After the composites were soaked in PBS for 1, 3 and 5 weeks, the proper weight ratio of GM was conformed by analyzing porosity, compressive strength and XRD. The morphologies of osteoblasts were examined with SEM after the cells were seeded on the CPC and GM/CPC for two days. After the cells were cultured with different leaching liquor of three kind of materials (0 wt%, 2.5 wt% GM/CPC and polystyrene) for days, cell viability and ALP concentration were measured. And 6 weeks after implantation into the bone defect of goats, GM/CPC and CPC were obtained and histopathological method was applied to investigate the bone ingrowth and the material degradation.3. The leaching liquor of rhBMP-2/CM/CPC or rhBMP-2/CPC was collected after both composites had been soaked in the Sodium Chloride for 1,4,7,14,21 and 28 days, and the concentration of rhBMP-2 was measured by ELISA method. Osteoblasts were cultured with both leaching liquor, and cell proliferation, ALP concentration and calcium nodule were investigated. Three weeks after implation in the muscle bags of the rats, the composites with the muscles around were collected and stained with hematoxylin and eosin; Ca and ALP concentrations of the muscles were also measured.4. Fifteen mature goats underwent ovariectomy and were placed on low cation relative to anion diet. After six months, bone defect of 5mm×10mm×10mm were made in L2,L4 of the goats, and three animals were not treated as blank control group. Others were randomly assigned to two groups and treated with rhBMP-2/GM/CPC (Group A) or rhBMP-2/CPC (Group B). After 1, 7 and 25 weeks, the defects were detected by CT scanning. Seven and twenty five weeks later, Animals were killed in batch, and BMD, compressive strength and micro-architecture of the vertebra were studied by dual energy X-ray absorption meter, biomechanical test and bone histomorphometry, respectively.Results1. GP and GA microspheres were both spherical with particle diameter of 78±18μm and 74±10μm, and there were no difference between both microspheres in drug loading and encapsulation rate; At the same crossling degree of 60%, GP microspheres, with longer degradation period (28 days) compared to GA microspheres (21 days), had better dispersibility, and swelling rate(89.0±4.8%),percentage of cumulative drug releasing in 10 days(78.8±4.96%)were both lower than GA microsphere(118.0±7.6%,90.5±5.12%); Cell cytotoxicity of 100%,50% and 25% leaching liquid of GP microspheres were all at theⅠlevel, but leaching liquid of GA microspheres with corresponding concentration were at the level ofⅢ,Ⅲ,Ⅱ.2. Porosity and macroporosity of the GM/CPC increased with the GMs increasing, but compression strength decreased after 1, 3 and 5 weeks of soaking, and the 2.5 wt% GM/CPC was the most favourable composite with high porosity and relative strong compressive strength. Osteoblasts showed normal morphology with both composites, but proliferation and differentiation of the cells were enhanced with the GM/CPC compared to the CPC. Both composites showed no adverse inflammatory reaction after weeks of implatation. New bones grew into the pores of the GM/CPC which resulted from GMs degradation, and coenocytes were present into the composite to degradate the material, but no new born could been seen inside of CPC.3. The release characteristics of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC both comprised two phases: (a) an initial burst occurring during the first 24 h, (b) a linear slow steady release phase linear release phase for the rest of the time course (days 2–28). Except for the similar initial burst effect, rhBMP-2 release rate of rhBMP-2/GM/CPC was faster than that of rhBMP-2/CPC in all the other evaluated time periods. In 28 days, 37.8±2.3% of the loaded protein in GMs was released, while in the control group the release rate was 14.7±1.9%. Cell experiment showed that Cell proliferation and ALP concentration of group A were significantly higher than those of group B, and calcium nodus of group A was bigger. In vivo studies also showed that quantity of cartilage formation, ALP and calcium concentration were higher in the group A.4. CT reconstrction analysis showd that bone defect healing was accelerated in the group A, and remain-volume of rhBMP-2/GM/CPC was smaller than that of rhBMP-2/CPC; in the untreated control group, a large defect could still be found in the vertebra after 25 weeks, although the bone defect became a little smaller. The results of biomechanical test showed that compressive strength of vertebra in the group A were 4.2±1.2 MPa for 7 weeks and 7.8±1.4Mpa for 25 weeks,which were higher than 2.4±0.9 Mpa and 4.6±0.8Mpa of the group B. Histomorphologically, trabecular thickness, relative trabecular volume and node/terminatio rate in the group A were higher than those in the group B, and trabecular separation was lower; more bone ingrowth was observed deeper into the rhBMP-2/GM/CPC composite, but no tissue formation was present inside the rhBMP-2/CPC composite; and almost half part of rhBMP-2/GM/CPC had degradated, but little resorption of rhBMP-2 /CPC could be detected. Mineralizition rate and bone density showed no difference between two groups after 7 weeks, but after 25 weeks the values were higher in the group A.Conclusion1.GMs crosslinked by GP have better biocompatibility and cotrolled-release ability than that crosslinked by GA, and GMs with crosslinking degree of 60% can be used as additive to construct macroporous CPC.2. Macroporous CPC can be developed using GP-crosslinked GMs, and 2.5 wt% GM/CPC is the most favourable with high macroporosity and strong compressive strength. The composite is a good bone substitute with improved degradability and biocompatibility.3.Controlled-release of rhBMP-2 and bone induction are both enhanced with rhBMP-2/GM/CPC compared to rhBMP-2/CPC. The new graft can accelerate healing of osteoporotic bone defect and improve bone quality significantly, therefore, it can be used as a promising bone substitute for bone defects of osteoporosis under non-loaded circumstances.