| Background:Bone fractures and bone defects are the most common diseases in orthopedic clinical practice, which are oftenly caused by high-energy trauma, osteoporosis and tumor erosion. Regarding to relatively simple fractures and bone defects, satisfying fracture healing can be easily achieved due to excellent healing potentials of native bone tissue and the assistance of internal instrumentation. However, with regard to complicated bone fractures (eg. comminuted bone fracture, CBF) and critical-sized bone defects, small bone fragments are difficult to fix merely by internal fixation devices and large bone defects are hard to bridge by relying solely on self-healing potential from host bone, which probably result in unsatisfactory results in bone repair. Therefore, developing novel bioactive bone substitutes are in urgent need. Injectable bone substitutes possess good fluidity and transformation ability to effectively hold bone pieces in place and bridge the gap between the bone interface in irregular bone defects and complex fractures, which make it widely accepted as promising candidates for those clinical applications.However, the application of most injectable bone substitutes, no matter clinically commercial ones or those in the animal study stage, are limited due to their own shortcomings in biomimiking natural bone repair processes. Polymethyl methacrylate (PMMA) bone cements and calcium phosphate cements (CPC), which are widely used in clinical practice, are non-biodegradable or low-biodegradable although they perform well in filling bone defects, stablizing bone fracture with strong mechanical strength. These shortcomings significantly inhibit bone regeneration and neovascularization. In contrast, the biodegradable hydrogel-based bone substitutes, which are still in experimental research stage, could provide much better biodegradation compared to PMMA bone cement and CPC. However, their adhesion strength to bleeding bone interface and osteogenetic ability (without incorporating osteoinductive proteins and growth factors) are far away from satisfaction. Therefore, new injectable and biodegradable bone substitutes exclusively for complex fracture are in need, which are supposed to possess following potentials:1) good surface adhesion strength in the oozing bone interface; 2) proper biodegradation rate to favor neovascularization and new bone ingrowth in scaffold; 3) osteogenic potential by incorporating osteoinductive small molecules into its chemical structure to promote bone regradation through degradation release.Herein, in order to meet the aforemention requiremtns, we incorporated citric acid, an small molecule with osteogenic potential, and dopamine, a strong adhesive molecule, into the chemical structure of the polymer through chemical bonds and developed a novel injectable bioadhesive bone substitutes, "injectable citrate-based mussel-inspired tissue bioadhesive -hydroxyapitite composite" (iCMBA/HA composite).As an important component of this polymer, citric acid has been found in recent studies that it plays essential roles in the regulation of bone formation and bone mineralization. Tran et al reported that citric acid can enhace alkaline phosphatase (ALP) activity and unregulate the expression of osteoblast-specific transcription factor (osterix, OSX). Hu et al found that citric acid is involved in regulating bone apatite nanocrystals thickness and apatite nanocrystals growth, as well as plays a vital role in maintaining the structure and mechanical properties of hydroxyapatite nanocrystals. Costello et al proposed that the process of osteoblasts "citation" in mineralization is closely associated to the alteration of metabolism of citric acid and believed that "citation" in osteoblasts, which means citric acid are over-produced in the cell metabolism and then secreted out of the cell to deposit in extracellular matrix, is an indispensable stage in the osteogenic differentiation process where stem cells are commited into osteoblast and then mineralized into osteocytes. All the latest research progress indicate that citric acid may plays an important role in the regulation of bone metabolism and bone formation and should be incorporated into the design of bone substitutes. L-3,4-dihydroxyphenylalanine (L-DOPA) is another important component of iCMBA polymer. As a bioadhesive molecule, it is originally found in secretions of aquatic animals among clamshell. Secretion of L-DOPA in aquatic animals make aquatic animals firmly attach to the wet surface under-water. This distinguished trait of L-DOPA gives us inspiration and push us to incorporate it into the polymer in order to enhance the adhesion strength of the bone substitute.In this study, we developed a novel biomimetic injectable bone substitutes (iCMBA/HA composites) and systematically characterized their physical and chemical properties, cyto-/bio-compatibility, osteogenicity in vivo and in vitro to verify the potentials mentioned above to optimize their formulations, providing important experimental evidence for large animal studies and clinical trials in the near future.Objective:1) To characterize chemical groups, physicochemical properties, mechanical properties and mineralization capabilities of iCMBA pre-polymer and iCMBA/HA composites in vitro; 2) To study the effect of degradation products of iCMBA/HA composites on osteoblast proliferation and differentiation in vitro; 3) To investigate the effect of iCMBA/HA composite on bone mass formation and bone quality restoration in comminuted radial fracture healing process in vivo; 4) To study role of citric acid in regulating the osteoblastic proliferation and differentiation.Methods:By using Nuclear Magnetic Resonance spectroscopy (NMR), infrared spectroscopy (FTIR), mechanical test and degradation release experiments, the chemical groups, physicochemical properties, mechanical properties and the in vitro mineralization ability of iCMBA prepolymer and iCMBA/HA composite were respectively characterized to optimize iCMBA/HA composite formulations. Next, the effect of the degradation products from iCMBA/HA composite on the proliferation of human mesenchymal stem cells (hMSC) and the proliferation of osteoblasts were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, Live/Dead cell viability test, alkaline phosphatase (ALP) activity assay, Western Blot and ELISA immunoblotting techniques in vitro. Then, iCMBA/HA70%(iCH70) bone substitutes were selected to treat comminuted radial fracture in New Zealand white rabbits and its potential of healing fracture and regenerating bone tissue were tested by Micro CT analysis, three point bending biomechanical test and histological analysis. At last, the regulation role of citric acid on osteoblastic bone marker protein (Runx2 and OCN) level were explored by Western Blot and Elisa immunoblotting techniques.Results:1. Characterization of iCMBA pre-polymer and iCMBA/HA compositeA) Characterization of chemical groups:Low-amplitude peak at 6.7 ppm (a) regions in nuclear magnetic resonance spectroscopy (1H-NMR) suggests that dopamine was successfully integrated into the side chain of iCMBA polymer backbone and the results were further confirmed by Fourier transform infrared spectra (FTIR) test.B) Characterization of set time:The set time of various iCMBA/HAcomposites ranged from 159±8 seconds to 247±13 seconds. The set time can be shortened by increasing the HA ratio in composites and enhancing PI concentration.C) Characterization of physical properties:The sol content of various iCMBA/HA composites were at low level, about 3%. The swelling ratio of iCH70 was about only 110%, which was significantly lower than that of iCH30 and iCH50 (P<0.05). Regarding to degradation rate, the degradation rate of iCH70 is relatively fast and complete degradation of iCH70 only need about 30 days.D) Characterization of mechanical properties:With the increase of hydroxyapatite (HA) content in iCMBA/HA composites, the compressive strength and Young’s modulus were enhanced. Once hydroxyapatite ratio increased to 70% (iCH70 group), the compressive strength of iCH70 reached up to 1.554±0.152 MPa, which are significantly higher than that of iCH50 and iCH30 group (p<0.05). Over time, the compressive strength would be increased to 2.592±0.253MPa and 3.182±0.272MPa, respectively.E) Characterization of In vitro mineralization:After incubation in simulated body fluid (SBF-5X) for one day, no calcium phosphate crystals were formed on the surface of iCH70 composites. Over incubation time, the calcium phosphate crystals begin to form and gradually grew and became obvious on Day 5. Energy Disperse X-ray (EDX) analysis validated the formation of calcium phosphate crystals and quantified the Ca/P ratio of calcium phosphate crystals, which was about 1:61. On the surface of pure iCMBA with no hydroxyapatite, no calcium phosphate crystal formation was observed.F) Characterization of accumulative citrate release:Citrate released in iCH70 degradation solution was 200uM on Day 2. The citrate release increased over time and reached to 900uM on Day 6. From Day 10 to Day 22, the citrate release reached a stable platform of 1200uM.2. In vitro experimentsA) Cytotoxicity test against hMSC:Sol content and degradation products of iCMBA/HA composite in each group showed low toxic to hMSC cells. The cell viability of hMSC in sol content solution maintained between 72.510±3.374% and 84.302±7.014%. The cell viabilities of hMSC in degradation products solution of each group were higher than that of PLGA (70.846±5.162%).B) Viability test against osteoblast:Osteoblastic activity in degradation product of iCH70 was high and few dead cells or apoptotic cells were observed. Osteoblast number increased over time. Their morphology distinguished from hMSC and began to aggregate to form calcium nodules.C) Osteogenic differentiation marker detection:The degradation products of iCH70 composite can upregulate Runt-related transcription factor-2 (Runx2) gene expression in early stage of osteogenic differentiation and enhance Alkaline phosphatase (ALP) activity in the middle stage and downregulate osteocalcin (OCN) expression levels in late stage of osteogenic differentiation.D) Osteoblastic mineralization detection:The number of calcium nodules formed in degradation solution of iCH70 increased over time and reached highest level on Day 14.3. In vivo animal experimentsA) Quantitative bone mass analysis by Micro CT scaning:No significant difference in bone mineral content (BMC) and bone mineral density (BMD) was seen between the iCH70 group and control group at the beginning day (P>0.05). After 4,8 and 12 weeks, the local BMC and BMD in iCH70 group were 387.167±9.827 mg and 122.000±5.586 mg/cm3,417.000±21.043 mg and 133.00±6.325 mg/cm3 427.000±17.413 mg and 143.00±8.579 mg/cm3, respectively. However, significant difference in BMC and BMD was observed only on Week 4 and Week 8 timepoints between the iCH70 group and control group (P<0.05).B) Three-point bending biomechanical test:At Week 4,8 and 12 timepoints, the Maximal Flexural load at radial fractures area in iCH70 group reached 128.000±3.688,146.000±3.521,178.500±4.848N, respectively, which were significantly higher than that of the control group (P<0.05).C) Bone Histological staining:The iCH70 composite degraded fast in vivo and were almost completely degraded in 30 days. Numerous vessles were formed in fracture healing area and hydroxyapatite particles scattering around were gradually incorporated into new bone area. After 12 weeks of iCH70 implantation, HA particles from composites were almost completely incorporated into the newly formed bone structure, which contribute to more organized bone structure and thicker cortical bone in iCH70 group.4. Effect of citric acid on osteoblastic proliferation and differentiationA) Safe Window of citrate concentration against hMSC proliferation:Low citrate concentration (20μM-2000μM) presented no inhibitory effect on hMSC proliferation; High citrate concentration (10000μM-20000μM) played significantly inhibitory effect on hMSC proliferation.B) Citrate regulation on bone marker expressionCitric acid (200μM) can upregulate Runt-related transcription factor-2 (Runx2) expression in early stage of osteogenic differentiation but could inhibit osteocalcin (OCN) expression in the late stage of osteogenic differentiation.C) Citrate role in osteoblastic mineralization:Citric acid (200μM) could promote the formation of calcium nodules in the extracellular matrix.Conclusion:Injectable citrate-based biodegradable bioadhesives-hydroxyapatite composites (iCMBA/HA composite) posess good injectability, proper degradation rate and mechanical strength, exellent cytocompatibility and osteogenic ability and are preferred candidate of injectable biodegradable bioactive bone substitutes exclusively for complex fractures such as comminuted bone fractures. More importantly, citric acid, the key degradation products of iCMBA/HA composites play indispensible role in regulating osteoblastic differentiation and mineralization process. |
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