Experimental Study About Fracture Healing-related Protein Expression Under The Influence Of New Absorbable PLLA/PLLA-gHA Material

Bone fracture is a common injury in the clinical practice of the orthopedics and cranio-maxillo-facial surgery. The healing of fractured bone is a complex physiological process, which includes vascular migration, angiogenesis and callus remodeling. Additionally, it involves the mediation of the differentiation and proliferation of cells by cell growth factor, as well as the effects of cell growth factor in the bone induction and molecular signal transduction. It is a multi-serial repairing process. In the management of patients with bone fracture, the traditional method is to use metal alloy as the internal fixer. The advantage of such material includes high mechanical strength and torsional modulus. However, it has the following limitations: The implantation of the metal fixer into the body may lead to electrolytic corrosion and subsequent release of metal ion. The later can cause inflammatory response of the local tissue, which thereby prompts a second surgery; the mismatch of bone rigidity and metal rigidity, which may lead to the absorption and weakening of the cortex bone underneath the internal fixation, thereby retarding the healing process. The high rigidity may increase the risk of another fracture of the reconstructed bone; T he metal internal fixer may induce the formation of fibrous cyst in the bone, which can separate the fixer and bone; The metal fixer must be removed after fracture healing to prevent bone atrophy (underneath the splint) due to lack of functional stimulation and inflammatory pain caused by the loosening and corrosion of metal instruments. Due to the limitations, many investigators have engaged in the development of absorbable bone fixation material. The emergence of bioabsorbable material allows investigators to establish a more accomplished internal fixation system. As the absorbable polymers materials do not require a second operation to remove, they are especially suitable for patients who only needs temporary implantation.The most extensively investigated material is poly Lactic acid (PLA); PLA is thermoplastic polyester and is made from fomented products or lactic acid of the plant stem and root. It is free of toxicity and stimulation, and has good bio-compatibility. The Food and Drug Admistration of USA labels it is a safe product. The mechanical strength is reduced by degradation, and stress is slowly shifted to the site of fracture, which can stimulate the healing process. Moreover, the metal covering effect during the use of metal fixer can be avoided, thereby, ameliorating psychological and physiological stresses of patients. The material can be completely degraded, absorbed and excreted by body. Thus, the second operation to remove the fixer is avoided. However, there are several limitations in its uses: i) the mechanical strength, rigidity and elastic modulus are all lower than natural bone cortex, thus, it can only be used for the non-weight bearing fracture sites. And the strength of such material needs to be improved. ii) Sterile inflammation may complicate the healing process during the material degradation.In an attempt to solve this problem, Chang Chun institute of applied chemistry Chinese academy of sciences developed the new PLLA/PLLA-gHA material (with independent intellectual property) for bone repairing. This technique has firstly performed grafting modification to gHA with the PLLA, the resultant complex PLLA-gHA is then mixed with PLLA to form the PLLA/PLLA-gHA mixture. HA is also called biological ceramics, which is similar to the bone matrix in the structure and composite, and has optimal tissue compatibility and effects of bone induction and conduction. We have conducted studies to assess the bio-compatibility of the mixture, the results showed that the PLLA/PLLA-gHA composite is free of cytotoxicity and acute toxicity that would not cause hemolytic reaction and rejection. It has biological safety and meets the relevant criteria. Compared with PLA material, it has higher mechanical strength and delayed degradation process, which satisfies the bone fracture fixation requirement of the beagle mandible. This material can improve the toughness to meet the mechanical requirement of fracture fixation. It can promote the adhesion and proliferation of the mouse embryo osteoblasts, and promote the osteoblasts to secrete extracellular matrix and form calcium nodules, which shows optimal compatibility with osteoblasts.With the development of molecular biology, various cell growth factors that have impact on the bone fracture healing have been discovered in recent years. And the understanding of fracture healing has transformed from histological level to the cellular and molecular levels. The initial phase of the study showed that, as compared with pure PLA material, PLLA/PLLA-gHA has optimal bone conduction property. However, the expressions of growth factors during the degradation of this material remain to be investigated. The growth factors are proteins of small molecular weight. Despite the small concentrations in the tissues and blood, the growth factors play important roles in the cell mitosis, matrix synthesis and tissue differentiation. The present study detected the expressions and distributions of TGF-β1, BMP2, bFGF, VEGF, IGF-I and PDGF by using immunohistochemical assay. And the expressions and distributions of typeⅠcollagen in callus were detected using Western blot method. The result will provide theoretical implications for clinical practice.The experiment was divided into three stages:(1) Beagle canine model of mandible fracture was established, which was fixed using PLLA/PLLA-gHA plate (experimental group) and titanium plate (control group). The canines were sacrificed at 1, 2, 3,4, 6,8 and 12 weeks after operation. The callus was obtained and sectioned for pathological examination. The sections were fixed, de-calcified, embedded and HE stained.(2) The modeling and sample harvesting were similar to the above-mentioned. The expressions and distributions of TGF-β1, BMP2, bFGF, VEGF, IGF-I and PDGF were detected by using immunohistochemical assay. The inter-group comparisons were performed to assess the mechanical property of PLLA/PLLA-gHA composite and its impact on the bone fracture healing.(3) The modeling and sample harvesting were similar to the above-mentioned. The expressions and distributions of CollageⅠin callus were detected using Western blot method.The results and conclusions of the study are as follows:(1) Compared with the experimental group and the control group, there was more fibroblasts increased in the experimental group, a large number of cartilage cells and osteoblasts proliferated, trabecular bone and angiogenesis appeared after 1 W; after 2W, endochondral bone formation in experimental group was more than in the control group, a large number of osteoblast proliferated, bone callus rich in blood vessels; after 3W: the experimental group was rich in osteoclasts and osteoblasts, new trabecular bone formed and cartilage formation was more than in the control group, bone salts deposited in cartilage matrix, angiogenesis was obvious, part of bone callus crossed the fracture gap, rich in blood vessels; after 4W, the formation of osseous callus and fibrous callus in the experimental group were more than the control group, the fracture gap was filled with new bone, more Haversian canal can be found; after 6W, osteoblasts and bone canaliculus in the experimental group was more than in the control group; after 8W, in the experimental group Haversian canal reduced in the fracture gap, new bone maturation appeared, bone cells arranged in concentric circles , bone lacunae arranged in neat rows, external callus is very small in amount; control group showed extensive bone formation, osteoblasts was in a random arrangement, fracture gap was connected by newly formed bone, The number of Lacuna is less than the experimental group; after 12W: fracture line disappeared and the difference between the experimental group and the control group decreased.(2) TGF-β1 protein was mainly expressed in osteoblasts, chondrocytes, and mesenchymal cells, the positive indexes in the postoperative experimental group after 2W, 3W, 4W were greater than the control group, and there was a significant difference (p <0.01 ), peak positive indexes in two groups appeared after 2W, then decreased gradually to no expression after 8w. Other time points no significant difference (p> 0.05).(3) BMP-2 protein was expressed in osteoblasts, chondrocytes, mesenchymal cells and bone matrix, peak positive indexes in two groups appeared after 2W, then decreased gradually to no expression after 12w. There was a very significant difference after 2W when compared the tow groups(p <0.01), after 3W, 4W there was a significant difference (p <0.05), the other time points no significant difference (p> 0.05).(4) bFGF protein was mainly expressed in osteoblasts, cartilage cells and bone matrix, the positive index began to rise since the 2w, 4w to peak, then decreased gradually to 12w when the negative expression showed. There was a very significant difference after 4W when compared the tow groups(p <0.01). Aafter 2W,3W there was a significant difference (p <0.05), the other time points no significant difference (p> 0.05).(5) VEGF protein mainly expressed in endothelial cells, cartilage cells, bone cells and osteoblasts, peak positive indexes in the two groups appeared after the 3W, then decreased gradually to no expression after 8w. Positive cell indexes in the experimental group after 2W, 4W, showed a significant difference than the control group (p <0.05), there was a very significant difference after 3W (p <0.01), the other time points difference was not statistically significant (p> 0.05).(6) PDGF protein was mainly expressed in mesenchymal cells, bone matrix, endothelial cells, cartilage cells and osteoblasts. In the experimental group after 1W, 2W,3W compared the two groups there was a significant difference (p <0.05),The peak value is located at 1W, the other time points no significant difference (p > 0.05).(7) IGF-1 protein mainly expressed in endothelial cells, osteoblasts and chondrocytes, peak positive index appeared at 2W, then decreased gradually to 12w when no expression appeared. Positive cell indexes in the experimental group after operation 1W, 2W, 3W were more than in the control group, which after 2W, 3W, compared the two groups there was a significant difference (p <0.01), after 1W there was a significant difference (p <0.05), the other time points no significant difference (p> 0.05).(8) Effect on type I collagen: the expression of type I collagen was weak in both groups after 1W, after 2W, expression levels in both groups increased, and the experimental group and the control group appeared significant difference (p <0.05). After 3W expression levels reached the peak in both groups and there was a significant difference between two groups (p <0.01). The expression of type I collagen in the two groups after4W was a significantly decreased, but the experimental group compared with the control group, there was still a significant difference (p <0.05). After 6W and 8W a slight expression of type I collagen appeared in both group, difference was not statistically significant (p> 0.05). There were no postoperative expression of type I collagen after 12W.Conclusions: the new bone repairing material PLLA/PLLA-gHA can satisfy the requirement of fracture fixation of the beagle mandible and can promote the healing of the mandible fracture of Beagles. Moreover, the protein expressions of TGF-β1, BMP2, bFGF, VEGF, IGF-I, PDGF and CollageⅠcan be significantly increased. The results provide biological basis for other basic researches of clinical application, thereby accelerating the early clinical use of the new material.