Osteoinduction with COLLOSS, COLLOSS E, and GFm.

This study provided data relevant to three major goals. It confirmed that both COLLOSS and COLLOSS E contain osteo- and chondro-inductive BMPs as shown by their ability to produce new bone in an ectopic location in rats. Second, based on the area of bone produced in standardized implant sections by osteoinductive growth factors in GFm, COLLOSS , and COLLOSS E and their respective collagenous carrier matrices, the study showed that COLLOSS was 0.1, and COLLOSS E 0.3 time as potent as 10 microg of GFm. Finally, the study showed that ordinary and accelerated endochondral bone formation were more frequent in response to GFm than to COLLOSS and COLLOSS E, whereas membranous bone formation was more frequent in response to COLLOSS E than to COLLOSS or GFm.

Non-glycosylated BMP-2 can induce ectopic bone formation at lower concentrations compared to glycosylated BMP-2.

Bone morphogenic protein-2 (BMP-2) is a well-known growth factor that can improve the biological performance of bone substitute materials. BMP-2 produced via bacterial expression systems are non-glycosylated (ng) whereas native and recombinant equivalents produced in mammalian cell expression systems are glycosylated (g) proteins. ngBMP-2 is less soluble, resulting in lower BMP-2 release from carriers as used as bone substitute materials. This seems promising for reducing the amount of included growth factor in bone substitute materials. Hence, it was hypothesized that ngBMP-2 would induce formation of the same amount of bone at an ectopic site at lower dosage as gBMP-2. To that end, gBMP-2 and ngBMP-2 were firstly in vitro comparatively evaluated for biological activity and release from a calcium phosphate (CaP) based bone substitute material. Thereafter, an ectopic implantation model in rats was used, in which gBMP-2 and ngBMP2 were loaded in various dosages (2-20 µg/implant) on the CaP-based bone substitute material and implanted for 4 and 12 weeks. The results revealed that both the in vitro biological activity of and the in vitro release of ngBMP-2 are lower compared to gBMP2. Upon ectopic implantation, however, ngBMP-2 loaded implants induced more bone formation at lower concentrations from 4-weeks onward compared to gBMP-2 equivalents, indicating the value of ngBMP-2 as a potential alternative for mammalian produced recombinant BMP-2 for bone regenerative therapies.

Healing of bone defects in the goat mandible, using COLLOSS E and beta-tricalciumphosphate.

COLLOSS E, an equine extracellular matrix product containing native transforming growth factor beta1 and several bone morphogenetic proteins, has shown osteoinductive properties in ectopic sites. This study was set up to examine its properties in an orthotoptic site in conjunction with a beta-tricalciumphosphate (beta-TCP) scaffolding material. Thirty-two 17-mm circular defects in goat mandibles were filled with COLLOSS E, beta-TCP, COLLOSS E + beta-TCP, or left empty. After 9 weeks the results were quantified by micro-computed tomography and histology. The empty defects contained the highest percentage of new bone (62%). The beta-TCP scaffold resulted in 38% (p = 0.0029), the mixture of beta-TCP/COLLOSS E resulted in 36% (p = 0.0057), while the use of COLLOSS E alone resulted in 55% (not significant p = 0.34). These results show that addition of TCP did not result in the expected synergy with regard to the healing of the defect and seemed even to inhibit the healing process. On the other hand, the addition of COLLOSS E induced the formation of small islands of new bone, not connected to the defect edges. This was not observed in the specimens not containing COLLOSS E (4.61% of bone formation centrally in the defect vs. 0.56%; p = 0.042). In conclusion, the results of the present study are somewhat unexpected in that the empty defects showed the most bone ingrowth; however, this ingrowth was always connected to the defect edges. In contrast, the application of COLLOSS E with or without beta-TCP induced bone formation in the center of the defects also.

Analytical assessment of the osteoinductive material COLLOSSE.

In this study, the growth factors in COLLOSSE were analyzed, using ELISA tests, mass spectrometry, western blotting, and a 24-day cell culture experiment using osteoblast-like cells. The results of the ELISA testing, mass spectrometry, and western blotting all confirmed that TGF-beta1 was the main growth factor in COLLOSSE at 55 ng/mg. The results from the culture test showed that the cell proliferation, alkaline phosphatase activity, and matrix calcification were all drastically changed by the addition of COLLOSSE, mirroring the effects of addition of TGF-beta1. We conclude that COLLOSSE is not only a rich source of TGFbeta-1, but also contains the growth factors TGFbeta-2, BMP-2, BMP-3, BMP-7, IGF-1, and possibly VEGF. Other growth factors might be present in COLLOSSE, but were not identified due to inherent detection limits of the used ELISA and mass spectrometry techniques. The number of osteoinductive factors in COLLOSSE causes a synergistic effect, explaining the new bone formation found in previously described in vivo studies, with much lower growth factor concentrations when compared with recombinant BMPs.

The effect of combined application of TGFbeta-1, BMP-2, and COLLOSS E on the development of bone marrow derived osteoblast-like cells in vitro.

This study investigated the combined application of Transforming Growth Factor beta-1 (TGFbeta-1) and Bone Morphogenetic Protein-2 (BMP-2) to stimulate osteogenic expression in vitro. TGFbeta-1 and BMP-2 fulfill specific roles in the formation of new bone. COLLOSS E, a bone-derived collagen product containing a variety of naturally occurring growth factors, was also used. Growth factors were administered to osteoblast-like cells from rat bone marrow (RBM). Proliferation and differentiation were monitored up to 24 days, by measuring total DNA content, alkaline phosphatase activity, and calcium content. Genetic expression of a set of differentiation markers at day 7 was measured by Q-PCR. Adding BMP-2 alone induced high proliferation rates, compared to the growth factor supplemented groups, and it induced high differentiation rates, compared to the control group. Adding TGFbeta-1 combined with BMP-2, TGFbeta-1 alone, or COLLOSS E resulted in a significant decrease in proliferation rate, but an increase in differentiation rate, compared to the control group. Additive or synergistic effects of application of TGFbeta-1 and BMP-2 were not observed. The observed effects of COLLOSS E mainly resembled those of TGFbeta-1 application alone. It can be concluded that BMP-2 is the most suitable candidate for osteogenic stimulation of RBM cells in these settings.