Canine cranial reconstruction using autologous bone marrow stromal cells

Abstract

Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects.

Figure 1

A: The dog calvaria prepared by creating equivalent, bilateral 35-mm bone defects. B: A BMSC-HA/TCP transplant was placed in one defect and a control, BMSC-free transplant placed in the other.

Figure 2

A: BMSC-free transplants 18 months postoperatively. Note extensive fibrovascular tissue with interspersed HA/TCP particles and only limited bone. B: BMSC-containing transplant 18 months postoperatively. Note extensive cortico-cancellous bone and hematopoietic adipose tissue. C: Higher power image of B. Note lamellar structure of bone, which is intimately associated with each particle. b, bone; f, fibrous connective tissue; p, particle, h, hematopoietic tissue.

Figure 3

Bone score as a function of time and transplant type. “All” includes transplants at time of harvest (6 to 7 months and 18 to 20 months) and biopsy (2 to 3 months). Error bars represent SD.

Figure 4

A: BMSC-free transplants 18 months postoperatively. Note extensive fibrovascular tissue, with little new bone forming a union with calvarium. B: BMSC-containing transplant 18 months postoperatively. Note appreciable zones of union between calvarium and new cortico-cancellous bone.

Figure 5

The extent of bone union as a function of time and transplant type. The data includes only transplants at time of harvest. Error bars represent SD.

Figure 6

A: CT image of dog head immediately after transplantation. qCT phantom is included with all examinations to provide density data. B: Six months after transplantation, the BMSC-containing transplant exhibits a greater density consistent with ossification, whereas the BMSC-free transplant exhibits a lucency consistent with HA/TCP particle resorption.

Figure 7

Scatter plot of bone score versus BMD for transplants at time of biopsy or harvest.

Figure 8

BMD as a function of transplant type, distinguished among transplants with poor bone formation (bone score 0 to 2) or good bone formation (bone score 3 to 4). Error bars represent SD.

Figure 9

Elastic (Young’s) modulus, as determined by noninvasive ultrasound evaluation. Elastic modulus E is uniformly greater among transplants with BMSCs. Error bars represent SD.

Figure 10

Elastic (Young’s) modulus of transplants, determined during ex vivo testing, at both their centers as well their margins. BMSC-containing transplants had uniformly higher E than their BMSC-free counterparts, whether measured at the center or margins of the transplants. A specific zone in one transplant (such as the center or margin of the transplant) is being compared to its counterpart on the contralateral transplant. Error bars represent SD.