Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain.

BACKGROUND: Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) "super-healer" mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant. METHODOLOGY: The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ "super-healer" mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints. PRINCIPAL FINDINGS: Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair. CONCLUSION AND SIGNIFICANCE: The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.

Plasticity of peptidergic innervation in healing rabbit medial collateral ligament.

BACKGROUND: Denervation substantially impairs healing of the medial collateral ligament (MCL). Because normal ligaments are sparsely innervated, we hypothesized that neuropeptide-containing neurons would sprout or proliferate after ligament transection, followed by later regression with healing, in a manner analogous to blood vessels. METHODS: We transected the right MCL in 9 mature female New Zealand white rabbits and killed 3 rabbits at 2, 6 or 14 weeks. Alternate sets of 12-mm serial sections of healing MCL scars were examined by fluorescent immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and pan-neuronal marker PGP9.5. RESULTS: Normal MCLs had few peptidergic fibres located in the epiligament in a perivascular pattern. At 2 weeks, PGP9.5-, SP-and CGRP-positive fibres had increased in the epiligament adjacent to the injury. By 6 weeks, there were increases in CGRP-and PGP9.5-positive fibres in epiligament and scar, with similar but less marked increases in SP-positive fibres. At 14 weeks, there was notable regression of immunostained peptidergic nerve fibres in the scar. CONCLUSION: This experiment shows evidence for a remarkable plasticity of ligament innervation after injury, supporting the idea that neuronal factors play a fundamental role in wound healing.

Nerve growth factor improves ligament healing.

Previous work has shown that innervation participates in normal ligament healing. The present study was performed to determine if exogenous nerve growth factor (NGF) would improve the healing of injured ligament by promoting reinnervation, blood flow, and angiogenesis. Two groups of 30 Sprague-Dawley rats underwent unilateral medial collateral ligament transection (MCL). One group was given 10 microg NGF and the other was given PBS via osmotic pump over 7 days after injury. After 7, 14, and 42 days, in vivo blood flow was measured using laser speckle perfusion imaging (LSPI). Morphologic assessments of nerve density, vascularity, and angiogenesis inhibitor production were done in three animals at each time point by immunohistochemical staining for the pan-neuronal marker PGP9.5, the endothelial marker vWF, and the angiogenesis inhibitor thrombospondin-2 (TSP-2). Ligament scar material and structural mechanical properties were assessed in seven rats at each time point. Increased nerve density was promoted by NGF at both 14 and 42 days. Exposure to NGF also led to increased ligament vascularity, as measured by histologic assessment of vWF immunohistochemistry, although LSPI-measured blood flow was not significantly different from controls. NGF treatment also led to decreased expression of TSP-2 at 14 days. Mechanical testing revealed that exposure to NGF increased failure load by 40%, ultimate tensile strength by 55%, and stiffness by 30% at 42 days. There were no detectable differences between groups in creep properties. The results suggest that local application of NGF can improve ligament healing by promoting both reinnervation and angiogenesis, and results in scars with enhanced mechanical properties.

Denervation alters mRNA levels of repair-associated genes in a rabbit medial collateral ligament injury model.

Previous experiments revealed that denervation impairs healing of the MCL. This suggested the hypothesis that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear or denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of 6 rabbits from each experimental group were killed. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbitspecific primers. In the denervated injury group, mRNA levels for the angiogenesis-associated gene MMP-13, matrix components Collagen I and III, growth factor TGF-beta and angiogenesis inhibitors TIMP-3, and TSP-1 had all increased by two-weeks post-injury, in comparison to the non-denervated injury group (p < or = 0.01). An increased level of TSP-1 mRNA was also detected in the denervated injured group at sixteen weeks post injury (p < or = 0.01). Contrary to the initial hypothesis, denervation led to increased mRNA levels for many relevant molecules during the early stages of MCL healing. Thus, inappropriate timing of over-expression of some molecules may potentially contribute to the decreased quality of the scar tissue, particularly molecules such as TSP-1. Neuronal derived factors strongly influence the in vivo metabolic activity of ligament and scar fibroblasts in the initial phases of healing.