Amelioration of proteoglycan accumulation in stress ...reverse transcription and real time...
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Amelioration of proteoglycan accumulation in stress-deprived tensile tendon of sheep shoulders by intralesional injection of heterologous bone marrow-derived mesenchymal stem cells. +Smith, M M; Ravi, V; Smith, S M; Hunyor, S; Dart, A J; Sonnabend, D H; Little, C B + 1 Raymond Purves Laboratories, Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW, Australia. [email protected] Introduction: Mesenchymal stem cells (MSCs) are being explored as a promising new treatment for many disorders, including tendinopathy [1]. These multipotent cells can, with the correct stimulus, differentiate into cells resembling those in cartilage, bone, fat and tendon. In our laboratories, an established sheep model of shoulder tendon injury induced by partial infraspinatus tendon transection allows the effects of both overstress (OS) and stress deprivation (SD) to be simultaneously evaluated in different regions of the one tendon [2]. Spatial and temporal changes in tendon pathology, collagen, proteoglycan and catabolic ADAMTS and MMP enzymes have been monitored for up to a year after induction of tendinopathy, with unresolved degeneration and eventual development of distinct foci of intratendinous chondroid metaplasia in both OS and SD tendon [3]. We recently demonstrated the feasibility of injecting MSCs into the surgically induced lesion, and now report the effects of such injections both acutely and chronically 3 months after induction of tendinopathy. Methods: Heterologous MSCs were isolated from sheep bone marrow, expanded and tested for multipotency (chondro- and osteo- differentiation). Eighteen 2 year-old Merino wethers had the infraspinatus tendon (IST) of one forelimb partially transected on the cranial side, midway between the humeral attachment and the musculotendinous junction. Six sheep served as non-operated controls (NOC). At 2 (acute n=6) or 11 weeks (chronic n=6) post-transection, 30 million MSCs in 0.25mL PBS were injected into the lesion site with ultrasound guidance. Thirteen weeks post-injury (i.e. 2 or 11 weeks after MSC injection), all IST were harvested and regions sampled from SD tendon either side of the transection (1: bone side and 3: muscle side) as well as from the lesion itself. Portions of each tendon region were formalin-fixed and processed into paraffin for histology. Sections (5µm) were stained with Toluidine blue, picrosirius red or H&E, and scored by 2 observers (MS & VR) blinded to treatment for: cell number, cell morphology, inflammatory infiltrates, vascularity, collagen fiber alignment and proteoglycan content [2]. Other portions of SD tensile tendon (region 3) were snap-frozen for subsequent total RNA extraction, reverse transcription and real time quantitative PCR using validated ovine-specific primers for ADAMTS4, ADAMTS5, ACAN, BGN, DCN, FMOD, LUM, VCAN, MMP3, MMP13, TIMP2 and TIMP3. Data was analysed for significance using Kruskal Wallis and Mann-Whitney U. Results: MSCs at the passage used for injection differentiated into chondrocytes and osteocytes in vitro, confirming their multipotency. The lesion was difficult to find with ultrasound at 11 weeks compared with 2 weeks post-injury, suggesting maturation of granulation tissue at this later timepoint. The gross morphology of the operated tendons was not visibly different between treatment groups; tendons were still shorter and thicker with enlarged and fibrosed epitenon. At 3 month post injury, histopathologic scores (Fig 1) for operated (Op) tendons were significantly higher than NOC in all regions (p<.003). Compared with Op sheep that had no injections, tendons injected with MSCs at 11 weeks post-Op. had significantly less pathology in region 3 (p=.045), worse pathology in region 1 (p=.0.041) and were not different in the lesion area. The improvement in region 3 pathologic scores was driven by significantly lower cellularity (p=.028) and cells having a more fibroblastic shape (p=.062). The tendons injected at the earlier timepoint trended to lower scores, reaching significance for cell shape only (p=.049). Interfasicular cellularity was significantly higher in lesion areas (p=.033) and region 1 (p=.041) but not in region 3 of Op tendons injected at 11 weeks post-Op. Proteoglycan scores (Fig 1 & 2) of Op tendons were significantly higher in the lesion area (p=.005) and region 3 (p<.001) but not region 1. Both tendons injected at 2 (p=.047) and 11 (p=.049) weeks had reduced accumulation of proteoglycan in region 3 but not in the lesion area. At 3 months post-injury, the significant increases (p<.05) in expression of MMP13, BGN, and LUM induced by SD in region 3 were not affected by MSC injection. DCN expression was significantly decreased by SD and was normalized by MSCs at 2 weeks. MSCs injected at 11 weeks reduced the expression of ADAMTS5, FMOD and TIMP3 expression. The expression of MMP1, ADAMTS4, ACAN, VCAN, TIMP2 were not affected by surgery or MSC injection in SD tensile tendon at 3 months. Figure 1. Histopathologic and proteoglycan scores for the lesion area and the adjacent SD tendon regions. *significantly different to Op, p<.05 Figure 2. Representative sections of region 3 tendon stained with Toluidine blue, showing changes in proteoglycan accumulation in injured tendon (proteoglycan is purple, cells are dark blue): A) NOC; B) Op; C) Op & MSCs at 2 weeks; D) Op & MSCs at 11 weeks. Discussion: Our previous experiments demonstrated that stem cells injected into a central lesion will localize in SD tensile tendon (region 3). Significantly, this is where we now see amelioration of proteoglycan accumulation two weeks after MSC injection. None of the changes seen in gene expression by MSC-injected tendons 3 months after surgically- induced tendinopathy explain the lowered proteoglycan content in region 3, suggesting a post-translational catabolic mechanism may be responsible, rather than a reduction in synthesis of these matrix molecules. In fact, the expression of the small proteoglycan DCN was significantly increased by the presence of MSCs. As this proteoglycan is involved in collagen structural alignment, collagen fiber remodelling may be enhanced. Release of trophic factors by the MSCs could invoke sequestration of enzyme inhibitors already present in the tendon matrix and /or activation of proenzymes, thus allowing existing but previously inactive or inhibited proteinases to increase the release of sulfated GAG. Nevertheless, we have uniquely demonstrated significant beneficial effects of injecting heterologous MSCs into pathological tendon in this animal model. References. [1] Chong AK, et al. (2009) Front Biosci 14:4598-605 [2] Smith MM, et al. (2008) Arthritis Rheum 58:1055-66. [3] Smith MM, et al. (2009) Trans ORS, 34:0029. Acknowledgements: Australian Orthopaedic Association and National Health and Medical Research Council of Australia for funding; Dan Burkhardt and Camden staff for animal logistics; Chris Ward for MSCs. Paper No. 11 • ORS 2011 Annual Meeting
Transcript of Amelioration of proteoglycan accumulation in stress ...reverse transcription and real time...
Amelioration of proteoglycan accumulation in stress-deprived tensile tendon of sheep shoulders by intralesional injection of heterologous bone marrow-derived mesenchymal stem cells.
+Smith, M M; Ravi, V; Smith, S M; Hunyor, S; Dart, A J; Sonnabend, D H; Little, C B
+1Raymond Purves Laboratories, Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW, Australia. [email protected]
Introduction: Mesenchymal stem cells (MSCs) are being explored as a promising new treatment for many disorders, including tendinopathy [1]. These multipotent cells can, with the correct stimulus, differentiate into cells resembling those in cartilage, bone, fat and tendon. In our laboratories, an established sheep model of shoulder tendon injury induced by partial infraspinatus tendon transection allows the effects of both overstress (OS) and stress deprivation (SD) to be simultaneously evaluated in different regions of the one tendon [2]. Spatial and temporal changes in tendon pathology, collagen, proteoglycan and catabolic ADAMTS and MMP enzymes have been monitored for up to a year after induction of tendinopathy, with unresolved degeneration and eventual development of distinct foci of intratendinous chondroid metaplasia in both OS and SD tendon [3]. We recently demonstrated the feasibility of injecting MSCs into the surgically induced lesion, and now report the effects of such injections both acutely and chronically 3 months after induction of tendinopathy. Methods: Heterologous MSCs were isolated from sheep bone marrow, expanded and tested for multipotency (chondro- and osteo-differentiation). Eighteen 2 year-old Merino wethers had the infraspinatus tendon (IST) of one forelimb partially transected on the cranial side, midway between the humeral attachment and the musculotendinous junction. Six sheep served as non-operated controls (NOC). At 2 (acute n=6) or 11 weeks (chronic n=6) post-transection, 30 million MSCs in 0.25mL PBS were injected into the lesion site with ultrasound guidance. Thirteen weeks post-injury (i.e. 2 or 11 weeks after MSC injection), all IST were harvested and regions sampled from SD tendon either side of the transection (1: bone side and 3: muscle side) as well as from the lesion itself. Portions of each tendon region were formalin-fixed and processed into paraffin for histology. Sections (5µm) were stained with Toluidine blue, picrosirius red or H&E, and scored by 2 observers (MS & VR) blinded to treatment for: cell number, cell morphology, inflammatory infiltrates, vascularity, collagen fiber alignment and proteoglycan content [2]. Other portions of SD tensile tendon (region 3) were snap-frozen for subsequent total RNA extraction, reverse transcription and real time quantitative PCR using validated ovine-specific primers for ADAMTS4, ADAMTS5, ACAN, BGN, DCN, FMOD, LUM, VCAN, MMP3, MMP13, TIMP2 and TIMP3. Data was analysed for significance using Kruskal Wallis and Mann-Whitney U. Results: MSCs at the passage used for injection differentiated into chondrocytes and osteocytes in vitro, confirming their multipotency. The lesion was difficult to find with ultrasound at 11 weeks compared with 2 weeks post-injury, suggesting maturation of granulation tissue at this later timepoint. The gross morphology of the operated tendons was not visibly different between treatment groups; tendons were still shorter and thicker with enlarged and fibrosed epitenon. At 3 month post injury, histopathologic scores (Fig 1) for operated (Op) tendons were significantly higher than NOC in all regions (p<.003). Compared with Op sheep that had no injections, tendons injected with MSCs at 11 weeks post-Op. had significantly less pathology in region 3 (p=.045), worse pathology in region 1 (p=.0.041) and were not different in the lesion area. The improvement in region 3 pathologic scores was driven by significantly lower cellularity (p=.028) and cells having a more fibroblastic shape (p=.062). The tendons injected at the earlier timepoint trended to lower scores, reaching significance for cell shape only (p=.049). Interfasicular cellularity was significantly higher in lesion areas (p=.033) and region 1 (p=.041) but not in region 3 of Op tendons injected at 11 weeks post-Op. Proteoglycan scores (Fig 1 & 2) of Op tendons were significantly higher in the lesion area (p=.005) and region 3 (p<.001) but not region 1. Both tendons injected at 2 (p=.047) and 11 (p=.049) weeks had reduced accumulation of proteoglycan in region 3 but not in the lesion area. At 3 months post-injury, the significant increases (p<.05) in expression of MMP13, BGN, and LUM induced by SD in region 3 were not affected by MSC injection. DCN expression was significantly decreased by SD and was normalized by MSCs at 2 weeks. MSCs
injected at 11 weeks reduced the expression of ADAMTS5, FMOD and TIMP3 expression. The expression of MMP1, ADAMTS4, ACAN, VCAN, TIMP2 were not affected by surgery or MSC injection in SD tensile tendon at 3 months. Figure 1. Histopathologic and proteoglycan scores for the lesion area and the adjacent SD tendon regions. *significantly different to Op, p<.05
Figure 2. Representative sections of region 3 tendon stained with Toluidine blue, showing changes in proteoglycan accumulation in injured tendon (proteoglycan is purple, cells are dark blue): A) NOC; B) Op; C) Op & MSCs at 2 weeks; D) Op & MSCs at 11 weeks.
Discussion: Our previous experiments demonstrated that stem cells injected into a central lesion will localize in SD tensile tendon (region 3). Significantly, this is where we now see amelioration of proteoglycan accumulation two weeks after MSC injection. None of the changes seen in gene expression by MSC-injected tendons 3 months after surgically-induced tendinopathy explain the lowered proteoglycan content in region 3, suggesting a post-translational catabolic mechanism may be responsible, rather than a reduction in synthesis of these matrix molecules. In fact, the expression of the small proteoglycan DCN was significantly increased by the presence of MSCs. As this proteoglycan is involved in collagen structural alignment, collagen fiber remodelling may be enhanced. Release of trophic factors by the MSCs could invoke sequestration of enzyme inhibitors already present in the tendon matrix and /or activation of proenzymes, thus allowing existing but previously inactive or inhibited proteinases to increase the release of sulfated GAG. Nevertheless, we have uniquely demonstrated significant beneficial effects of injecting heterologous MSCs into pathological tendon in this animal model. References. [1] Chong AK, et al. (2009) Front Biosci 14:4598-605 [2] Smith MM, et al. (2008) Arthritis Rheum 58:1055-66. [3] Smith MM, et al. (2009) Trans ORS, 34:0029. Acknowledgements: Australian Orthopaedic Association and National Health and Medical Research Council of Australia for funding; Dan Burkhardt and Camden staff for animal logistics; Chris Ward for MSCs.
Paper No. 11 • ORS 2011 Annual Meeting
