Enhancement of the repair of meniscal wounds in the red-white zone (middle third) by the injection of bone marrow cells in canine animal model

Abstract

Bone marrow stem cells (BMSCs) can differentiate into several cells that participate in the healing of meniscal wounds. To test this hypothesis, we examined the effects of injected BMSCs on the healing of meniscal wounds. Autologous BMSCs from eight adult dogs were injected into meniscal wounds (knee joints). After 12 weeks, the healing process was clinically and immunomorphologically evaluated using: (i) histochemical stains (haematoxylin and eosin, Masson trichrome and periodic acid-Schiff) and (ii) immunoperoxidase staining methods (CD3, CD79a, CD68, CD31 and alpha smooth-muscle actin for T, B lymphocytes, macrophages, endothelial cells and smooth-muscle lineage). Complete (six vs. three), partial (one vs. one) and no healing (one vs. four animals) of the meniscal wounds were observed in the injected and noninjected menisci. As compared with the noninjected menisci, examination of the tissues from the injected ones revealed: (i) marked angiogenesis (microvessel density: 3.22 +/- 0.66 vs. 6.50 +/- 2.10); (ii) chondrogenesis; (iii) prominent immune cell infiltrate (4.07 +/- 0.78 vs. 9.56 +/- 1.69, 8.33 +/- 0.77 vs. 3.67 +/- 1.00 and 4.38 +/- 0.62 vs. 11.1 +/- 1.43 for the total numbers of immune cells, lymphocytes and macrophages, respectively); and (iv) proliferation of the fibroblasts with marked deposition of collagen fibres (2.0 +/- 0.84 vs. 2.66 +/- 0.48). These values were statistically significantly higher for the injected menisci as compared with the noninjected ones (P >/= 0.05). Autologous BMSCs can improve meniscal wound healing. Whether this improvement occurs through BMSC differentiation into cells operational in the repair process, the release of certain mediator or other unknown mechanisms mandates further investigations.

Figure 1

(a) Bone marrow was aspirated from the posterior superior iliac spine of the dog's iliac bone by using a spinal needle; (b) following bone marrow centrifugation, four layers were evident representing plasma, mononuclear cells, Ficoll–Hypaque and packed red blood cells, respectively; (c) the bone marrow was injected into the meniscal tissues at the wound site; (d) clotted bone marrow was injected into the meniscal wound; (e) above-knee cylindrical cast and (f) complete healing of the meniscal wound without evidence of previous tears.

Figure 2

(a, b) Angiogenesis manifesting as several capillary sized blood vessels in the healed meniscal tissues following the injection of the bone marrow stem cells [(a) haematoxylin and eosin and (b) Masson trichrome x400]; (c, d) lack of angiogenesis in the noninjected menisci [(c) haematoxylin and eosin and (d) Masson trichrome ×400].

Figure 3

(a) Marked deposition of strongly stained collagen fibres [haematoxylin and eosin (H&E) ×400]; (b) prominent immune cell infiltrate (H&E ×1000); (c) chondrogenesis (PAS ×400) in the healed meniscal tissues following injection of bone marrow stem cells; (d) mild deposition of faint collagen fibres (H&E ×400); (e) sparse immune cell infiltrate (H&E ×1000) and (f) lack of chondrogenesis (H&E ×400) in the noninjected menisci.

Figure 4

(a) Immunoperoxidase staining for CD79a (B lymphocytes); (b) CD3 (T lymphocytes); (c) CD68 (macrophages); (d) CD31 (endothelial cells) and (e) alpha smooth-muscle actin (smooth muscles of the blood vessels) in the healed meniscal tissues following their injection with bone marrow stem cells (×1000).