| Natural bone regeneration/healing process is orchestrated by a spectrum of growthfactors, which have been found to be essential for onset and progression of the initialhealing response and are believed to be the key to accelerate healing and tissueregeneration.Delivery of a combination of growth factors or their encoding genes has tosome extent stimulated bone healing in vivo. However, this type of exogenous stimulationmay interrupt the bone and blood vessel remodeling balance, leading to the excessive boneformation spreading outside the original contour of the defect or hypotension andconsequent vascular leakage, besides the technology hurdles to deliver multiple factorssimultaneously in a controlled manner. Moreover, efficient viral gene delivery is generallyaccompanied with the risk of tumorigenesis or unwanted adverse immuneresponses.Therefore, the potential of extrogenous stimulation to enhance growth factors(GFs) is often impaired. Osteoporosis, the most frequent bone remodeling disease, is caused by a relativeincrease of bone resorption over bone formation. Approximately one in two women andone in four men over the age of40are expected to suffer from osteoporotic fracture in theworldwide.Although a variety of therapeutics have been used in clinics for the treatmentof osteoporosis, the vast majority are anti-resorptive agents which exert their effect bydecreasing the rate of bone resorption. Less attention has been paid on the strategy aimingat the reversal of reduced bone formation and re-building of damaged bone tissues. Assuch, only parathyroid hormone (PTH) has been approved by FDA to be used in clinics tostimulate bone formation with intermittent injection period up to2years. However, thetreatment may increase bone resorption and the continuous daily administration cost toomuch for the2-year period. There is a need for the research into new molecular that mightlead to the development of novel anabolic therapeutics targeting bone formation process.miRNAs, the noncoding~22-nucleotide RNAs serving as repressors of gene expressionat the post-transcriptional regulation level, are involved in a broad spectrum of biologicalprocesses.The founding members of the miRNA class, lin-4and let-7were discovered inC. Elegans to regulate the developmental timing and progression of the nematode lifecycle. To date,1424miRNAs have been identified in human cells. Computationalpredictions indicate that each is predicted to regulate several target genes.More than50%of all human protein-coding genes are potentially regulated by miRNAs. An increasingnumber of miRNAs has been identified to regulate osteoblast differentiation and boneformation positively by targeting negative regulators of osteogenesis or negatively bytargeting important osteogenic factors. Overexpression or inhibition of miRNAs canregulate the endogenous expressions of multiple growth factors simultaneously. It wastherefore hypothesized that delivery of a desired miRNA may result in optimization ofbone regeneration through coordinating of endogenous angiogenesis and osteogenesisprocess.miRNAs play critical roles in diverse biological and cellular processes includingmetabolism,differentiation, and apoptosis. Aberrant miRNA expression has beenimplicated in the pathogenesis of a large number of human diseases including cancer,diabetes, neurological disorders, heart failure, pulmonary hypertension and autoimmune diseases due to dysfunction of their target genes expression in human disorders. miRNAexpression profiling may be useful as diagnostic or prognostic tools and can help intreatment decisions in cancer and other diseases.miRNAs have several significant advantages to be developed as new therapeutic agentsin that they are safe, efficacy, small and comprise of a known sequence that is oftencompletely conserved among species, which are very attractive features from a drugdevelopment standpoint. However, potiential application of using miRNA-based strategiesin the treatment of human disease remains in their infancy. Multiple challenges such asoptimization of selectivity, stability, delivery and long-term efficiency, have to beaddressed in order for miRNA treatment to become a successful therapeutic agents.Based on the background above, we firstly comprehensively analyzed the reportedosteogenesis-angiogenesis relative miRNAs expression profile through the computationalprediction strtegy and identify the miRNA (miR-26a) with the maximum capacity toregulate angiogenesise-osteogenesis coupling. Then we found the enhanced endogenousmiR-26a expression in the defect area could lead to optimized bone repair. Furthernore,we found that miR-26a overexpression in the OVX-MSC could rescue its impaired boneformation ability both in vitro and in vivo. To continuously deliver miRNA to the specificsite of the body and thereby enhance key osteogenic factors expression at therapeutic levelfor prolonged time, We developed an high efficiency, low toxicity and biologicaldegradable miRNA transfection complex and further constructed an controlled releasedsystem to locally deliver the complex for a desired time with full bioactivity to achievedthe optimized clinical effect. We found that the cell-free strategy beased miRNA deliverysystem leaded to optimized bone repair through simutanouly sustain multipleosteogenesis-angigenesis key factors st at high level for prolong time. The promisingresults indicated that the cell-free miR-26a continuously delivery system could be atherapeutic candidate to enhance bone formation.Results1Overexpression of miR-26a in BMMSCs promoted its osteogenic function through upregulating osteogenesis-angiogenesis coupling in vitro and further enhance its ectopicbone formation ability in vivo.Furthermore, we contructed an critical size calvarial bonedefect and implanted with the in vivo miRNA delivery system We found that the enhancedendogenous miR-26a expression at the defect and surrounding tissue significantlyenhanced both vascularization and bone formation. The effects were mainly due toincreases expression levels of endogenous key factors involved in different osteogeneticand angiogenetic developmental in vivo；2miR-26a expression level was found to be negatively correlated with bone loss inosteoporotic mice through the real time RT-PCR test. Overexpression of miR-26a inOVX-MSC significantly enhanced Alp, Ocn expression and thereby rescued the reducedthe Alp activity and impaired mineralization ability of OVX-MSC.Futhermore,overexpression of miR-26a in OVX-MSC has effectively rescued the impaired boneformation ability of OVX-MSC in vivo；3. We synthesized the biodegradable miRNA transfection system with low toxicity andhigh transfection efficiency via polymerzation and click chemistry based on polyether andpolyester. To sustain the desired miRNA (miR-26a) delivery for a desired time with fullbioactivity to achieved the optimized clinical effect, we entrapped the polylex into thePLGA microsphere and thereby it can be continuously delivered to function on targetgenes for prolong time. To deliver the desired miRNA selectively to the specific site of thebody, we attached the PLGA microsphere on the nanofibrus PLLA scaffold so that it canbe locally delivered to the healing area. The cell-free system we established was found tooptimize enhancing the bone repair utilizing host regeneration capacity.Conclusion1Overexpression of miR-26a in BMMSCs promoted osteogenic function throughupregulating osteogenesis-angiogenesis coupling in vitro. Enhanced endogenous miR-26aexpression at the defect and surroundingtissue significantly enhanced both vascularizationand bone formation ability in vivo； 2Overexpression of miR-26a in OVX-MSC significantly rescued the reduced Alp activityand impaired mineralization ability of OVX-MSC in vitro.Futhermore, Overexpression ofmiR-26a in OVX-MSC has effectively rescued the impaired bone formation ability ofOVX-MSC in vivo；3The miRNA locally controlled release system we contructed could overcome thedifficulties of using miRNA-based strategies in the treatment of disease.It couldcontinuously deliver the desired miRNA to function on target genes for prolong time andthereby lead to optimized bone repair without cell seeding on it. |
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