Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2.

Masako Fujioka-Kobayashi,Richard J Miron,Nikola Saulacic,Benoit Schaller,Yufeng Zhang,Benjamin E Pippenger

doi:10.1002/cre2.55

Abstract

The use of growth factors for the regeneration of soft and hard tissues has been utilized extensively in dental medicine over the past decade. Recently our group found that recombinant human bone morphogenetic protein 9 (rhBMP9) was more osteopromotive than recombinant human bone morphogenetic protein 2 (rhBMP2) when combined with a deprotenized bovine bone mineral bone grafting material. The aim of the present in vitro study was to evaluate the regenerative potential of an absorbable collagen sponge(ACS) specifically designed for extraction socket healing loaded with rhBMP9 when compared to rhBMP2. The adsorption and release kinetics of rhBMP2 and rhBMP9 were first investigated by enzyme‐linked immunosorbent assay quantification. Then, the cellular effects of stromal cell line (ST2) preosteoblasts were investigated utilizing four groups including rhBMP2 and rhBMP9 at both low(10 ng/ml) and high(100 ng/ml) concentrations loaded onto ACS. Cellular attachment(8 hours) and proliferation(1, 3, and 5 days) as well as osteoblast differentiation were investigated by real‐time polymerase chain reaction (PCR) at 3 and 14 days, alkaline phosphatase (ALP) activity at 7 days, and alizarin red staining at 14 days. ACS fully adsorbed both rhBMP2 and rhBMP9 that were slowly released up to 10 days. Although neither rhBMP2 nor rhBMP9 had any effects on cell attachment or proliferation, pronounced effects were observed on osteoblast differentiation. ALP activity was increased seven‐fold with rhBMP2‐high, whereas a marked 10‐fold and 20‐fold increase was observed with rhBMP9‐low and high loaded to ACS, respectively. Furthermore, mRNA levels of collagen1, ALP, bone sialoprotein, and osteocalcin were all significantly higher for rhBMP9 when compared to control or rhBMP2 groups. Alizarin red staining further confirmed that rhBMP9‐low and high demonstrated marked increases in mineralization potential when compared to rhBMP2‐high. The results demonstrate the marked effect of rhBMP9 on osteoblast differentiation when combined with ACS in comparison to rhBMP2 at doses as much as 10 times lower. Further in vivo studies are necessary to investigate whether the regenerative potential is equally as potent.

Highlights

AbstractThe use of growth factors for the regeneration of soft and hard tissues has been utilized extensively in dental medicine over the past decade
absorbable collagen sponge (ACS) loaded with recombinant human bone morphogenetic protein 9 (rhBMP9) high (100 ng/ml) demonstrated significantly higher alkaline phosphatase (ALP) staining when compared to all other groups representing up to a 20‐fold significant increase when compared to control samples and a 2.5 fold significant increase when compared to recombinant human bone morphogenetic protein 2 (rhBMP2) at the same concentration (100 ng/ml; Figure 3)
The aim of the present study was to investigate the biocompatibility of ACS and their ability to promote osteoblast differentiation when combined with rhBMP2 and rhBMP9

Summary

| MATERIAL AND METHODS

Recombinant human BMP2 and rhBMP9 were purchased from R&D systems Inc (Minneapolis, MM, USA). In order to determine the quantity of rhBMP2 and rhBMP9 protein being released from ACS over time, coated ACS were soaked in 1 ml of PBS, and samples were collected at various time points including 15 min, 1 hr, 8 hrs, 1, 3, and 10 days. ST2 cells were stimulated on ACS for 14 days with/without rhBMP in osteogenic differentiation medium, which consisted of DMEM supplemented with 10% FBS, 1% antibiotics, 50 μg/ml ascorbic acid (Sigma) and 10 mM β‐glycerophosphate (Sigma) to promote osteoblast. After days, cells were fixed in 96% ethanol for min and stained with 0.2% alizarin red (Alizarin red S; Sigma) solution in water (pH 6.4) at room temperature for 1 hr as previously described (Fujioka‐Kobayashi et al, 2016a). The effects of rhBMP2 and rhBMP9 had no influence on the proliferation of preosteoblasts at either 1, 3, or 5 days postseeding (Figure 2b)

| RESULTS

| DISCUSSION

Findings

CONFLICT OF INTEREST