In vitro characterization and in vivo assessment of equine tendon-derived progenitor cells

Durgam, Sushmitha S.

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https://hdl.handle.net/2142/95260 Copy

Description

Title

In vitro characterization and in vivo assessment of equine tendon-derived progenitor cells

Author(s)

Durgam, Sushmitha S.

Issue Date

2016-08-30

Director of Research (if dissertation) or Advisor (if thesis)

Stewart, Matthew C.

Doctoral Committee Chair(s)

Stewart, Matthew C.

Committee Member(s)

• Wagoner Johnson, Amy J.
• Berry-Miller, Suzanne E.
• Fan, Timothy

Department of Study

Vet Clinical Medicine

Discipline

VMS-Veterinary Clinical Medcne

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Tendon-derived stem cells
• Tendon healing

Abstract

Tendinitis is a common cause of breakdown injury in equine athletes and accounts for 30% to 50% of all racing injuries. The last decade has seen significant development in mesenchymal stem cell (MSC)-based therapies in tendon repair. The focus on tendon-derived progenitor cells (TDPCs) for tendon healing is based on the rationale that stem cells obtained from tendons are more phenotypically-committed or ‘primed’ for tenogenesis than cells from other tissues. The overall objective of this body of research is to characterize and evaluate equine tendon-derived progenitor cells for tendon healing in horses. TDPCs were isolated via a differential adhesion preplating screen that has been successfully used to isolate skeletal muscle-derived stem cells. Cell suspensions obtained via collagenase digestion of equine lateral digital extensor tendon (n=4) were serially transferred into adherent plates every 12 hrs for 4 days. TDPCs obtained from the initial, third and seventh preplates were used for subsequent analyses. Growth/proliferation and basal tenogenic gene expression of the three TDPC fractions were largely similar. Preplating and subsequent monolayer expansion did not alter the immunophenotype (CD29+, CD44+, CD90+, and CD45−) and trilineage differentiation capacity of TDPC fractions. Overall, TDPCs were robustly osteogenic but exhibited comparatively weak adipogenic and chondrogenic capacities. These outcomes indicate that preplating does not enrich for tendon-derived progenitors during in vitro culture, and ‘whole tendon digest’-derived cells are as appropriate for cell-based therapies. In vitro growth characteristics of matched equine TDPCs and bone marrow MSCs (BM-MSCs) during monolayer expansion were assessed (n=6). Subsequently, third passage TDPCs and BM-MSCs were cultured on acellular tendon matrices for 7 days with or without insulin-like growth factor supplementation. Matrix production and matrix gene expression were analyzed at the end of in vitro culture. During monolayer expansion, at each passage, the yield of TDPCs was 3-fold higher than the matched BM-MSCs. The viability of TDPCs on acellular tendon matrices was 1.6-2.8 fold higher than BM-MSCs. New collagen and glycosaminoglycan syntheses were significantly greater in TDPC groups and in IGF-I–supplemented groups. The mRNA concentrations of collagen type I and III, and cartilage oligomeric matrix protein (COMP) were not significantly different between TDPC and BM-MSC groups. These in vitro results demonstrated that TDPCs may offer a useful resource for cell-based therapies for tendon healing. Lastly, the efficacy of TDPCs in an in-vivo equine flexor tendinitis model was evaluated. Collagenase-induced tendinitis was created in both front superficial digital flexor (SDF) tendons (n=8). Four weeks later, the forelimb tendon lesions were treated with 1 x 107 autogenous TDPCs or saline. Twelve weeks after forelimb TDPC injections, tendons were harvested for assessment of matrix gene expression, biochemical, biomechanical and histological characteristics. Collagen I and III, COMP and tenomodulin mRNA levels were similar in both TDPC and saline groups and higher than normal tendon. Yield and maximal stresses of the TDPC group were significantly greater than the saline group’s and similar to normal tendon. However, the elastic modulus of the TDPC and saline groups were not significantly different. Histological assessment of the repair tissues with Fourier transform-Second Harmonic generation imaging demonstrated that collagen alignment was significantly better in TDPC group than in the saline controls. In summary, TDPC administration improved the histological and biomechanical properties of collagenase-induced tendinitis lesions.

Graduation Semester

2016-12

Type of Resource

text

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Copyright and License Information

Copyright 2016 Sushmitha Durgam

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