Search

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 μg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.

Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits.

Osteoinductive characteristics of new osteoplastic materials based on demineralized bone matrix of xenogenic origin with high and controlled degree of purification were studied on the model of regeneration of critical-size cranial defects in rats using modern approaches, including histological analysis, evaluation of morphological parameters of the bone tissue obtained by micro-computed tomography, and estimation of bone tissue growth rate using in vivo fluorochrome label. Demineralized bone matrix and, to a much greater extent, its activated form containing modified recombinant growth factor rhBMP-2 with high content of the dimeric form exhibited osteoinductive activity.

Comparative analysis of serum cytokine profiles of CBA mice was carried out 1, 5, 24, and 48 h after intraperitoneal injection of killed culture of different streptococcus A types. The production of cytokines in response to different streptococcus types varied. The highest level was recorded in response to types 1M and 3T+M, more often detected in invasive streptococcal infection. The highest levels of IL-2 were recorded in response to 1M (47-fold increase in comparison with the control) and 3T+M streptococcus types (more than 10-fold increase). Injections of these types also led to an increase of IFN-γ level (15.6 and 11.3 times, respectively). The level of TNF-α increased less (3.6 times in response to 3T+M and 2.6 times in response to 1M type). The levels of IL-5, IL-10, and IL-12 increased 2-3-fold. Injections of 1T and 5M types led to just a 2-fold increase of cytokine levels. These data indicated induction of the immune response trend by mainly Th1 or mixed Th1/Th2 pattern in response to group A streptococcus antigens.

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P(+) colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P(+) colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was significantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1β, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.

Test system ELISA-BMP-2 is developed for measuring recombinant human bone morphogenetic protein-2 in human and laboratory animal serum and plasma by sandwich ELISA. The test system has been used for studies of the kinetics of bone morphogenetic protein-2 release from collagen carrier in the presence of plasma proteins.

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.

Immunization of CBA mice with killed group A streptococcus (type 5) vaccine changed the counts of stromal precursor cells (CFC-F) in bone marrow transplants at different donor-recipient combinations (normal, N, or immune, I). CFC-F counts in bone marrow transplants from normal mice transplanted to immunized animals decreased 4-6-fold depending on the transplant age in comparison with similar transplants in normal recipients. The percentage of CFC-F colonies with alkaline phosphatase (osteogenesis marker) activity decreased more than 2-fold. Similarly, the count of CFC-F in the transplants was 2-fold lower during delayed (7 months) period after bone marrow transplantation from immunized donors (8-12 days after the end of immunization) to intact recipients, while 2 months after transplantation it was 3-fold lower. The mean optical density of the bone capsule in preparations stained for glycogen and alkaline phosphatase was 1.5-3 times lower in the N-->I and I-->N experiments in comparison with the control (N-->N). On the other hand, CFC-F count in the femoral bone marrow of immunized animals was significantly (3.5-2.5 times) higher during the period from 8 days to 8 months after the end of immunization compared to CFC-F count in the femoral bone marrow of intact mice. These results attest to a significant prolonged effect of streptococcal antigens on the bone marrow stromal tissue. These data also indicate that not all CFC-F, the counts of which increased in response to antigens, are responsible for transplantability of the stromal tissue in heterotopic transplantation. Immunization by streptococcal antigens seemed to suppress transplantability and osteogenic activity of stromal stem cells. The efficiency of CFC-F cloning in mouse bone marrow cultures increased significantly (2-3-fold) in the presence of sera from immune mice. The levels of TNF-α and IFN-γ were low in this serum (2.7 and 6 times lower, respectively) in comparison with normal serum. Presumably, the effects of streptococcal antigens on stromal tissue were mediated through serum cytokines.

The efficiency of cloning of stromal precursor cell in mouse bone marrow culture increases significantly (2-3-fold) in the presence of serum from mice immunized with type 5 group A streptococcus antigens (5-20 microl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-alpha and IFN-gamma are significantly reduced (2.7 times and more than 6-fold, respectively) in the sera of immunized mice in comparison with normal serum. Serum levels of IL-2, -4, -5, -10, and -12 were about the same in both groups; no granulocyte-macrophage CSF was detected. These data attest to appreciable effect of immunization with streptococcal antigens on the bone marrow stromal tissue; this effect is presumably mediated through serum cytokines.