We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically.

Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (sd 4.5) versus 12.7% (sd 2.9, p < 0.019), respectively.

Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.

Bone growth was compared in six types of (beta-tricalcium phosphate) implants implanted in subcutaneous pouches or close to femoral head of male Wistar rats:

implants immersed in 0.9% sodium chloride solution (control implants),

Implants were removed 21 days after operation and dissected following principles of stereology. Presence of bone or cartilage or connective tissue was evaluated by hematoxylin eosin histochemistry.

Results: Bone formation was only found in the implants where BMP-2 was introduced. However, no distinctive differences were found between the implants where cells and BMP-2 were introduced and between the implants where just BMP-2 was used. Percentages of the bone tissue out of all the implant were as follows: 0.0% in group 1, 1.2% in group 2, 32.4% in group 3, 42.4% in group 4, 44.4% in group 5 and, 54.9% in group 6. Differences in amount of bone tissue were statistically significant between groups 3 and 2, groups 3 and 1 and also between groups 1 and 2 (p=0.0013, p=0.0004 and p=0.0525 respectively). In the other cases, the differences between BMP-2 affected implants and implants without BMP-2 were even greater.

We concluded that presence of osteoconductive matrix and introduction of an osteoinductive agent (e.g. BMP-2) are the main components of designing of bone tissue and introduction of exogenous bone cells is not as important as the first two in subcutaneous pouches or close to the hip joint.

Ex vivo cell-growing technique might be a solution for treatment of bone diseases leading to the local bone defects. We assessed the effect of ex vivo-cultured cells in ectopic bone induction in animals with normally functioning connective tissue cells.

Material and methods: Bone marrow cells, harvested via puncture of tibial canal of male Wistar rats, were cultured, and differented into osteogenic lineage using chemical stimulus.

After differentiation osteoprogenitor cells were transferred into beta-tricalcium phosphate scaffolds using either centrifugation or simple diffusion. Six types of implants (beta-tricalcium phosphate matrixes) were implanted into subcutaneous pouches. In the first group saline-immersed implants were used; in the second group the ex vivo cells were transferred into the implant by diffusion and in the third group by centrifuging; in the 4th, 5th and 6th group the implants were processed as in first three groups, respectively, but 12.5 microgram of rhBMP2 was added to the each implant. After 21 days the implants were removed and dissected systematically. Histomorphometry analysis was performed following the principles of stereology.

Results and discussion: Bone formation was found only in the rhBMP2-immersed implants. Other implants consisted mostly of connective tissue and in lesser extent of the unchanged scaffold. No distinctive differences were found between the rhBMP2-implants. The osteoinduction seems to be crucial in ectopic bone formation if there is no cellular dysfunction present. The inductive effect of rhBMP2 cannot be compensated by the abundance of the pre-differentiated osteogenic cells as shown by the absence of bone induction in the groups two and three in this model.

• Supported by Estonian Government SF 0180030s07