RESUMO
Heterotopic ossification (HO) is a serious disorder that occurs when there is aberrant bone morphogenic protein (BMP) signaling in soft tissues. Currently, there are no methods to detect HO before mineralization occurs. Yet once mineralization occurs, there are no effective treatments, short of surgery, to reverse HO. Herein, we used in vivo molecular imaging and confirmatory ex vivo tissue analyses of an established murine animal model of BMP-induced HO to show that matrix metalloproteinase-9 (MMP-9) can be detected as an early-stage biomarker before mineralization. Ex vivo analyses show that active MMP-9 protein is significantly elevated within tissues undergoing HO as early as 48 h after BMP induction, with its expression co-localizing to nerves and vessels. In vivo molecular imaging with a dual-labeled near-infrared fluorescence and micro-positron emission tomography (µPET) agent specific to MMP-2/-9 expression paralleled the ex vivo observations and reflected the site of HO formation as detected from microcomputed tomography 7 days later. The results suggest that the MMP-9 is a biomarker of the early extracellular matrix (ECM) re-organization and could be used as an in vivo diagnostic with confirmatory ex vivo tissue analysis for detecting HO or conversely for monitoring the success of tissue-engineered bone implants that employ ECM biology for engraftment.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Ossificação Heterotópica/diagnóstico , Ossificação Heterotópica/enzimologia , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Camundongos , Imagem Molecular , Dados de Sequência Molecular , Imagem Multimodal , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Tomografia por Emissão de Pósitrons , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Tomografia Computadorizada por Raios XRESUMO
Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.