Abstract
Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide-co-glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11–34% for GDF5 and 6–17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.
Highlights
Substantial progress has recently been achieved concerning new synthetic biomaterials for bone repair and replacement, e.g., polymers, metals, ceramics, bioactive glasses, calcium sulfates, calcium carbonates, and calcium phosphates [1]
Clinical efficacy of vertebroplasty/kyphoplasty has been reported for both PMMA cement [21] and calcium phosphate cement (CPC) [22,23], for CPCs there are some concerns on the long-term loss of the correction of the vertebral kyphosis angle [24,25]
The different bone morphogenetic proteins (BMPs) analyzed in the present study showed differential induction of alkaline phosphatase (ALP) activity in individual marker cell lines—i.e., GDF5 and BMP-2 reacted with all cell lines except for C2C12—whereas the GDF5 mutant BB-1 reacted with all cell lines
Summary
Substantial progress has recently been achieved concerning new synthetic biomaterials for bone repair and replacement, e.g., polymers, metals, ceramics, bioactive glasses, calcium sulfates, calcium carbonates, and calcium phosphates [1]. On the basis of promising previous in vitro and in vivo studies with a biodegradable, brushite-forming CPC with reinforcing PLGA fibers [7,8,9,10,11,12], the present study is focused on this particular mineral–organic bone replacement composite. Calcium phosphate cement (CPC), first described in the 1980s [13,14], represents a potential alternative to polymethylmethacrylate (PMMA) for the surgical treatment of osteoporotic vertebral compression fractures, due to its biodegradability, fast setting ability, and osteointegrative capacity [15]. The elastic modulus of CPC (180 MPa, similar to that of cancellous bone) may avoid stress shielding effects and abnormal load transfer [20], potentially reducing secondary fractures of adjacent vertebral bodies due to the much higher elastic modulus of PMMA (2700 MPa). Clinical efficacy of vertebroplasty/kyphoplasty has been reported for both PMMA cement [21] and CPC [22,23], for CPCs there are some concerns on the long-term loss of the correction of the vertebral kyphosis angle [24,25]
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