Hydrogels and DMP1 delivery: a strategy to improve bone reconstruction procedures. In vivo study in osteoporotic rats

• Grant number: 25/02133-5
• Support Opportunities: Regular Research Grants
• Start date: August 01, 2025
• End date: July 31, 2027
• Field of knowledge: Health Sciences - Dentistry - Oral and Maxillofacial Surgery
• Principal Investigator: Roberta Okamoto
• Grantee: Roberta Okamoto
• Principal researcher abroad: ANNE GEORGE
• Institution abroad: University of Illinois at Chicago (UIC), United States
• Host Institution: Faculdade de Odontologia (FOA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil
• Associated researchers: Karina Midori Mori Carneiro

Abstract

The increase in population longevity leads to an increase in the prevalence of metabolic diseases, including osteoporosis, while at the same time causing an increase in the demand for oral rehabilitation involving osseointegrated implants. It is important to highlight that these rehabilitations, in many cases, require reconstructive bone procedures. Patient's systemic condition is one of the factors that needs to be considered in the process of bone reconstruction; and osteoporosis is one of the conditions that can harm them. Considered a public health problem in postmenopausal women due to the depletion of estrogen production, osteoporosis is characterized by a decrease in bone mass and imbalance of bone metabolism, which can compromise the success of rehabilitation involving osseointegrated implants. In this context, the functionalization of biomaterials with bioactive molecules is a strategy that can improve bone reconstruction procedures in patients with tissue involvement. Thus, this study aims to investigate the impact of local release of DMP1 protein using two different hydrogels on critical calvaria defects of osteoporotic rats. We will take an interdisciplinary methodological approach to this challenge by using an osteoporotic rat model, created by bilateral ovariectomy (OVX) surgery, predisposed to osteoporosis. Firstly, the in vitro stage of the study will be carried out, in which, after the functionalization of DNA and GelMa hydrogels with DMP1, cell culture will be carried out to determine cell viability, micronucleus testing and alkaline phosphatase activity, in addition to the study of UV/VIS to determine the release rate of DMP1. Subsequently, in a second study, the in vivo characterization will be carried out. To this end, the impact of hydrogels functionalized with DMP1 on collagen production and its inflammatory potential will first be investigated. For this, 4 experimental groups composed of normal physiological rats (SHAM) will be used, which will have DNA or GelMa hydrogels, functionalized with or without DMP1, implanted in the dorsal subcutaneous (SC) region (I) SC SHAM GelMa; (II) SC SHAM GelMa + DMP1; (III) SC SHAM DNA and (IV) SC SHAM DNA + DMP1. The animals will be euthanized at 3, 7 and 10 days post-operatively. Histological analyzes (hematoxylin and eosin and picrosirius red staining) and immunohistochemistry (RUNX2, ALP, OPN, OCN, TRAP, OPG and RANKL) will be performed. Subsequently, the impact of local release of DMP1 through different hydrogels on critical calvaria (CA) defects in osteoporotic rats will be investigated. For this study, eight experimental groups, osteoporotic (OVX) or control (SHAM), will receive GelMa or DNA hydrogels, functionalized with or without DMP1: (V) CA SHAM GelMa; (VI) CA SHAM GelMa + DMP1; (VII) CA SHAM DNA (VIII); CA SHAM DNA + DMP1; (IX) CA OVX GelMa; (X) CA OVX GelMa + DMP1; (XI) CA OVX DNA and (XII) CA OVX DNA + DMP1. The animals will be euthanized at 10, 28 and 60 days post-operatively. We will characterize the structural, cellular and molecular response using a multi-modal combination of histologic (hematoxylin and eosin and picrosirius red staining) and immunohistochemical - RUNX2, ALP, OPN, OCN, TRAP, OPG and RANKL (10, 28 and 60 days), micro-computed tomography (micro-CT - BV. TV, Tb.Th, Tb.N, Tb.Sp, Po.tot and I.S) (28 and 60 days), fluorochrome labelling for bone apposition - mineral apposition, active mineralization surface and bone dynamics (28 days), and real-time polymerase chain reaction (RT-PCR - RUNX2, ALP, iBSP, OCN, TRAP, OPG and RANKL) for gene expression identification (10 days). This study will greatly impact the current understanding of how DMP1, a protein responsible for initiating the nucleation of hydroxyapatite in vivo and initiating the biomineralization process, would impact bone reconstruction in the face of osteoporosis, given that, to date, it has never been applied in vitro or in vivo in the face of this systemic condition. (AU)