| BACKGROUNDThe most important functions for skeleton system of a human are to maintain the body movement, support the weight and protect organs. Trauma, infection, tumor, debridement for osteomyelitis and congenital diseases are the main causes of the bone defect. Conservatively estimated, hundreds of billions dollars were consumed on the treatments of orthopedic diseases in USA. It was the same in China that numerous patients spent their wealth on bone transplantations. Autogenous, allogeneic, artificial bones are common materials for bone tissue regeneration at present. Autogenous bones transplantation is the most commonly used therapy now and also considered as the gold standard to treat bone defects. There are many advantages of autogenous bones transplantation, such as no rejection, free from contamination, economical, absorbable, inductive osteogenesis. Allogeneic bones benefit form more abundant sources compared to autogenous bones, besides, low immunogenicity and structural support. But insufficient bone volume, additional incision to bone harvesting, extension of anesthesia and operation time, relatively more complications, long-time pain or discomfort around the bone harvesting site are shortages of autogenous bones transplantation. Similarly, possible immunological rejection and infection are deficiencies of allogeneic bones transplantation. Artificial bones can only be bone defects filler and supports not induce osteogenesis. The most commonly used artificial bones are calcium phosphate cement and acrylate cement. But their biocompatibility, mechanical strength, osteoconduction still cannot meet the demand of clinic. Therefore, how to regenerate bone defects better is always a hot spot in tissue regeneration research.Concerning the biomaterial research, novel bone implant materials, bone tissue engineering and application are becoming the most popular research in orthopaedics gradually in recent years. Scaffold is the core element in bone tissue biomaterial research. It can simulate the bone defect morphology and be a shell frame that connecting cells and tissue so that tissue will be induced to regenerate into specific shape. Meanwhile, scaffold plays a mechanical support role to resist extraneous pressure as well as a place for cells to proliferation, differentiation and metabolism. With the developments of biomaterial engineering, research in biomaterial scaffold is increasing richly. The most researched biomaterial scaffolds at present are calcium phosphate cement (CPC), tricalcium phosphate composite (TCP), polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA). All of the biomaterial scaffolds above were reported to have different advantages in osteogenesis. Fabricating bone tissue substitute biomaterials in vitro that similar to the natural bone in constituent and structure is considered as an effective method to overcome the shortages in bone tissue substitute biomaterials mentioned above. Tricalcium phosphate (TCP) is a common constituent in human bone.β-tricalcium phosphate is the most configuration used in biomaterials for its favorable biocompatibility, biological activity and biological degradability.3-tricalcium phosphate may be an ideal regeneration and substitute biomaterial for human bone tissue. β-tricalcium phosphate have been wildly used in orthopaedic and stomatology tissue regeneration.Relative bone tissue engineering basic research so far have established abundant theories and technical methods to fabricate biofunctional bone substitute materials that combining seed cells, biological active factors and biomaterial scaffold. Mesenchymal stem cell (MSC) is multipotent stem cell from mesoderm. It can be found mainly in connective tissue and organ mesenchyme, especially higher level in bone marrow tissue. Bone marrow mesenchymal stem cell (BMSC) is one of the most studied stem cells now due to its advantages, such as favorable ability of multi-directional differentiation and easy to be obtained. BMSC, as one kind of seed cells, have been wildly researched in cell and organ damages in the fields of heart tissue, liver tissue, bone tissue, cartilage tissue. Related research showed that different source of stem cell leads to different reaction to a same biomaterial because of their inherent differences and different source of the stem cell had different effect in osteogenesis.It was considered that scaffold, seed cells, cytokines are indispensable three elements in bone tissue regeneration biomaterial. But with the development of biomaterial research, study in cytokines become rich. Cytokines become more and more important in biomaterial research. Various kinds of biomaterials combined with cytokines showed varying degrees of advantages in promoting osteogenesis and angiogenesis. So cell-free scaffolds combined with cytokines provide a new clue in the field of biomaterial research. The mostly common used cytokines are bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF). BMP-2 is one of the transforming growth factor-β(TGF-β) superfamily members. It is able to promote osteogenesis and differentiation of osteoblast, at the same time, make them express the specific osteogenic products. PDGF-BB is an important mitogenic factor. It is capable to promote proliferation of osteoblast. A mass of PDGF-BB, which can make the osteoprogenitor cell into osteoblast, are generated in local bone fracture. So that the healing of bone fracture will be promoted.The osteogenic and angiogenic effects of bone substitute biomaterials affected by different cytokines are still in dispute. Therefore, it become more and more meaningful to enhance the bone substitute biomaterial research, develop more novel biofunctional bone substitute biomaterials combined with cytokines, meanwhile, observe the degradation rate, bone conduction and osteogenic function. This is not only significant to increase the awareness of the bone substitute biomaterials but also provide some brand new treatment thoughts and technical methods for clinic bone defects.OBJECTIVETo harvest the bone marrow mesenchymal stem cells from SD rats. Then cultivate and proliferate bone marrow mesenchymal stem cells in vitro. After that, co-culture with β-tricalcium phosphate scaffold. Observe the bone marrow mesenchymal stem cell of SD rat’s proliferation and differentiation on novel β-tricalcium phosphate composite. Furthermore, fabricate a β-TCP scaffold combined with BMP-2/PDGF-BB to regenerate SD rat’s cranium critical-size bone defects. To observe the regeneration effect of the new-type bio-functional biomaterials in SD rat critical-size cranial defects and collect experimental data of basic research, osteogenesis and angiogenesis in bone substitute biomaterials.METHODS1.Bone marrow mesenchymal stem cell of SD rat’s proliferation and differentiation on novel β-tricaicium phosphate compositeThe biomaterial CaPfromGem21 was fabricated into some 3.6mm in diameter, 1.5mm in thickness discoid scaffolds. Mesenchymal stem cells were isolated from bone marrow of SD rat’s both tibias and fibulas by density gradient centrifugation and cultivated in vitro. Flow cytometry detection was performed on passage 3 to ascertain the surface markers of the cells. The scaffolds were placed individually in 24-well plates. Then BMSCs in passage 3 was seeded dropwise on the top of the β-TCP scaffold at 3×105 cells per scaffold and cultivated with osteogenic media contain 100 nM dexamethasone,10 mM p-glycerophosphate,0.05 mM ascorbic acid, and 10 nM 1a,25-dihydroxyvitamin. As for control group, the BMSCs were cultured with normal medium. After 1,4,7,14 and 21 days, scanning electron microscope was performed according to the experiment plan, cell viability was assessed using the live/dead viability/cytotoxicity kit. The expression levels of the collagen type I (CI), Runx-2, osteocalcin (OC) and ALP genes were quantified by the quantitative real-time polymerase chain reaction technique (qRT-PCR). The alkaline phosphatase (ALP) activity and mineralization of BMSCs in both groups were also measured.2.β-Tricaicium phosphate composite pre-loaded with bone morphogenetic protein-2 or platelet derived growth factor-BB for critical-size cranial defects regeneration in SD rats.The biomaterial used in this research is P-tricalcium phosphate called Calcium Phosphate from Growth-factor Enhanced Matrix 21(CaPfromGEM21) that preloaded with Bone Morphogenetic Protein-2(BMP-2) or Platelet Derived Growth Factor-BB (PDGF-BB). Each group contained BMP-2 or PDGF-BB. The contents of the growth factors are:BMP-2(10ng/100ng), PDGF-BB (0.3ug/3ug). In addition, there are one control group (without any factor) and one blank group, six experiment groups in total. All the implants are grouped as followed:(1) CaPfromGEM21/control; (2) CaPfromGEM21+10ng BMP-2; (3) CaPfromGEM21+100ng BMP-2; (4) CaPfromGEM21+0.3ug PDGF-BB; (5)CaPfromGEM21+3ug PDGF-BB; (6)Blank. The animal used are three-month old SPF level male Sprague-Dawley rats. All the rats are anesthetize by pentobarbital intraperitoneally. Two 3.6mm in diameter full-thickness critical-size defects were made in the cranium of each rat and different implant groups were filled in each defect randomly. Then sacrificed all the animals 6 weeks after surgery. Detection methods for the samples include X-ray, micro CT, hematoxylin-eosin (HE) staining and related quantative analysis.ResultsSD rat’s bone marrow mesenchymal stem cell showed a strong growth in vitro. Flow cytometry indicated that the cells’surface markers matched those of mesenchymal stem cells. Generally observation, SD-BMSC maintain continuous proliferation on the scaffold. Scanning electron microscope detection showed that BMSC attached on the β-TCP scaffold, stretched fully and naturally. Live/Dead staining showed a same active proliferation. The percentage of live cells indicated that there was no significantly difference of two groups at each time point. Viable cell density assessment showed that the viable cell density was increasing over time in both group. The density of 7/14 days were significantly higher than 1/4 days. There was no significantly difference between two groups at the same time point. ALP activity measurements showed that the activity of osteogenic group increased over time while control group showed no obvious rising trend. The ALP activity of osteogenic group on 14/21 days were significantly higher than other time points. QRT-PCR showed that the osteogenic genes of all experiment groups reached their peaks on different time points. The expression of ALP gene peaked at day 4, about 5-folds to that on day 1. The expressions of COL-1 and OC gene both peaked at day 7, about 6-fold to that on day 1. And RunX-2 in gene expression reached peak on day 14,3-fold to that on day 1. Meanwhile, the control group showed no obvious peaks. The alizarin red staining showed that the surface color deepened over time. Further mineral synthesis quantify analysis indicated that the mineral quantity on day 21 was significantly higher than that on day 14 and day 7 of osteogenic group, and significantly higher than that of the control group at the same time points.There is neither accidental death because of anesthesia during the operation nor infection post-operation. The X-ray images indicated all the defects were repaired in different extents. The fracture line became indistinct and PDGF-BB group were almost fully regenerated. Micro CT showed new bone formation in the defects. The quantative analysis showed that new bone volume of the PDGF-BB groups were significantly more than the BMP-2 groups (P<0.05). HE staining showed no acute or chronic inflammatory cells but new bones in the defects and new vessels with red blood cells inside in and around the new bones. The new bone area fraction quantative analysis indicated that lOng BMP-2 group and 0.3ug PDGF-BB group were significantly more than CaPfromGEM21 control group (P<0.05). The new vessel density analysis indicated that four cytokine-preloaded groups were significantly more than blank group and CaPfromGEM21 control group (P<0.05), but there were no significate different between four cytokine-preloaded group (P> 0.05).ConclusionThe novel β-tricalcium phosphatescaffold is hypotoxic, biocompatible. BMSC can attach on its surface and keep a rapid proliferation and satisfying osteogenic potential. CaPfromGem21 combined with BMP-2 or PDGF-BB remained satisfying biocompatibility. The regeneration effects in cytokine-preloaded groups were better than control group. These kinds of biomaterial might become perfect bone defect regeneration materials. |
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