Bone Regeneration Using Biomimetic Scaffold Material Based On Mineralized Collagen Combined With A Synthetic BMP-2-related Peptide

PartⅠEffect of BMP-2-related peptide on osteogenic differentiation andmineralization of BMSCs cultured in three-dimensional systemObjective To evaluate the effect of BMP-2-related peptide on osteogenic differentiationand mineralization of BMSCs cultured in three-dimensional systemMethods BMSCs were harvested from 4-week-old SD rats and cultured with normalmedium containing low-glucose Dulbecco's modified Eagle's medium (DMEM), 10% fetalbovine serum (FBS) and 1% antibiotics. The 3rd generation BMSCs were dissociated.collected and cultured with Cytodex^TM 3 collagen microcarrier in three-dimensional system.After one day, the medium was changed to osteogenetic differentiation medium whichconsisted of the culture media previously described, supplemented with 10mMβ-glycerophosphate and 50 mg/ml ascorbic acid. In the experimental group, the medium wasadded to 200μg/ml BMP-2-related compared with the control group. After 7d and 14d. thecell survival of BMSCs was determined by a fluorescent live/dead assay. The calciumcontents and alkaline phosphatase(ALP) staining were measured to assess the differentiationof BMSCs towards osteoblasts. The mineralization of BMSCs was determined by calceinstaining. Results BMSCs cultured with Cytodex^TM 3 collagen microcarrier grew well in bothexperimental group and control group. There was no statistical significance between the twogroups (P＞0.05).The result of calcium contents and ALP staining suggested theexperimental group was significant higher than the control group(P＜0.05). The result ofcalcein staining showed that the mineralization level of BMSCs in the experimental groupwas significant higher than that in the control group.Conclusion BMP-2-derived peptide can promote osteogenic differentiation andmineralization of BMSCs cultured in three-dimensional system effectively.PartⅡPreparation and characteristic of scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptide andits effect on biological behavior of bone marrow stromal cellsObjective In this study, a new biomimetic bone tissue engineering scaffold material, thescaffold based mineralized collagen loaded with BMP-2-related peptide was prepared. Theeffect of BMP-2-related peptide on the adhesion, proliferation and differentiation of BMSCswere investigated.Methods The scaffold material nano-hydroxyapatite/collagen/poly(lactic acid)(nHAC/PLA) was developed by biomimetic synthesis.The hierarchical microstructure of thescaffold material was analyzed by X-ray diffraction. The microcosmic appearance of thescaffold was observed by scanning electron microscope. BMP-2-related peptide wasintroduced into the porous nHAC/PLA scaffold. The third generation of BMSCs was seededonto the scaffolds and the scaffold without BMP-2-related peptide as a control. The adhesionof BMSCs was assessed by precipitation method. The proliferative ability of BMSCs weremeasured by MTT assay and scanning electron microscope. The alkaline phosphatase activities and calcium contents were measured to assess the differentiation of BMSCstowards osteoblasts.Results The result of X-ray diffraction suggested that the inorganic phase in themineralized collagen scaffold was determined as HA. The results of scanning electronmicroscope showed that the surface of the scaffold was porous and the pores wereinterconnected. The pore sizes were from several 10μm to about 300μm. The abilities ofadhesion and differentiation to osteoblast of the BMSCs in the experimental group weresignificantly greater than those in the control group (P＜0.05), but with regard to the abilitiesof proliferation of the BMSCs. there is no significant difference between the experimentalgroup and the control group(P＞0.05).Conclusion The synthetic BMP-2-related peptide could improve biocompatibility andbioactivity of the nHAC/PLA scaffold material. The nHAC/PLA scaffold loaded withBMP-2-related peptide is a kind of ideal scaffold material for bone tissue engineering.PartⅢEctopic osteogenesis of scaffold based on mineralizedcollagen loaded with synthetic BMP-2-related peptideObjective To evaluated the ectopic osteogenetic capacity of the scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptide.Methods The scaffold material nano-hydroxyapatite/collagen/poly(lactic acid)(nHAC/PLA) was prepared. BMP-2-related peptide was introduced into the porousnHAC/PLA scaffold. For observing the releasing character of BMP-2-related peptide.thereleased peptide content from the scaffold was detected using high performance liquidchromatography at different set times. 48 SD rats were allocated into four groups randomly.Four kinds of scaffold materials were respectively implanted subcutaneously into rats. Group A: the nHAC/PLA scaffold loaded with 3 mg BMP-2-related peptide, Group B: thenHAC/PLA scaffold loaded with 2 mg BMP-2-related peptide, Groups C: the nHAC/PLAscaffold loaded with 1 mg BMP-2-related peptide, Groups D: the nHAC/PLA scaffold alone.At the 4th and 8th weeks after implantation, the rats were sacrificed in batch and the sampleswere harvested. Their osteogenic capability was detected by CT scan and histologicalobservation.Results The releasing character of BMP-2-related was that an initial burst releasing(29.57%) was observed over the first day, followed by a sustained release and reached 65.98% after 21 days. The results of CT scan and histological observation demonstrated that theosteogenic capability of 3 mg, 2 mg and 1 mg of the BMP-2-related peptide was superior tothe implants without the BMP-2-related peptide(P＜0.05). There was no significant differencebetween implants with 3 mg and 2 mg BMP-2-related peptide(P＞0 05), but the osteogeniccapability of the two dosage groups was significantly better than that of the 1 mggroup(P＜0.05).Conclusion BMP-2-related peptide can increase the osteoinduction of nHAC/PLAscaffold and the BMP-2-related peptide induced ectopic bone formation in a dose-dependentmanner. The nHAC/PLA scaffold loaded with the BMP-2-related peptide is a kind of idealscaffold material with good osteoinductivity.PartⅣRepair of cranial bone defects with scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptideObjective To investigate the osteogenetic capacity of nHAC/PLA loaded with aBMP-2-related peptide biomimetic scaffold materials.Methods Rat cranial defects were created on both sides of the parietal bone of 24 adult Sprague-Dawley rats. BMP-2-related peptide combined with nHAC/PLA scaffold wereimplanted on the left side as experimental group, leaving the other side implanted withnHAC/PLA scaffold alone as control group. Up to the 4^th and 12^th weeks, the rats were killedin batch respectively. Macroscopic observation, three-dimensional reconstruction of computedtomography, histological observation and scanning electron microscope observation wereperformed on these samples. Biomechanics detection, Masson trichome staining, andimmunohistochemical analysis were performed on the 12^th week's sample additionally.Results Three-dimensional reconstruction of computed tomography indicated thatthere were radiodense areas in the left dorsal regions of each rat. but the density of theradiodense areas of experimental group were obviously denser than the density of controlgroup at the 4^th week. The defects completety healed at the 12^th week in the experimentalgroup that was treated with BMP-2-related peptide loaded nHAC/PLA, while the completebone healing rate in control group was just 1/2. Histological observation showed a largeamount of osteoid tissue and new bone had been observed in the experimental groups at the4th week. While the control group showed only minimal new bone formation at the defectmargins. At the 12th week, in the experimental group, the bony-union between new bone andhost bone was observed. Meanwhile. the composite was almost completely degraded. In thecontrol group, there were still slight amounts of new bone, the scaffolds were only partlydegraded and the residual materials were surrounded by areas of new bone formation. Thepercentage of bone formation areas in the experimental group was superior to the controlgroup at 4^th and 12^th weeks, the differences were statistically significant (P＜0.05). Scanningelectron microscope demonstrated that the bone defects were repaired completely in theexperimental group, where most composite was degraded and replaced by lamellar bone,compared with partly repaired in the control group. Masson trichome staining at the 12^th weekshowed that massive calcified bones stained red color were observed in the experimentalgroup. However, in the control group there was only slight amounts of fresh-formed bonestained blue color. The results of immunohistochemical staining for osteocalcin (OCN) and osteopontin (OPN) further supported the H&E and Masson trichrome staining findings.Positive OCN and OPN staining were found throughout the overall region of the regeneratedbone in the experimental group. In contrast, the regenerated tissue from the control groupshowed much lower OCN and OPN immunoreactivity than the experimental group.Biomechanics detection indicated the maximum compression loading and loading/strain ratioon repairing tissue of defects in the experimental group was obviously superior to those in thecontrol group. The differences were also statistically significant (P＜0.01)Conclusion The osteogenic capability of BMP-2-related peptide loaded nHAC/PLA isobviously superior to nHAC/PLA alone. BMP-2-related peptide loaded nHAC/PLAbiomimetic scaffold material can be a potential repairing material for bone defects.