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Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

Mienaltowski MJ, Adams SM, Birk DE - Stem Cell Res Ther (2014)

Bottom Line: Tendon construct ultrastructure was also compared after 45 days.It also was found that peritenon-derived progenitors secrete factor(s) stimulatory to tenocytes and tendon proper progenitors.Data demonstrate that, relative to peritenon-derived progenitors, tendon proper progenitors have greater potential for forming functional tendon-like tissue.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Multipotent progenitor populations exist within the tendon proper and peritenon of the Achilles tendon. Progenitor populations derived from the tendon proper and peritenon are enriched with distinct cell types that are distinguished by expression of markers of tendon and vascular or pericyte origins, respectively. The objective of this study was to discern the unique tenogenic properties of tendon proper- and peritenon-derived progenitors within an in vitro model. We hypothesized that progenitors from each region contribute differently to tendon formation; thus, when incorporated into a regenerative model, progenitors from each region will respond uniquely. Moreover, we hypothesized that cell populations like progenitors were capable of stimulating tenogenic differentiation, so we generated conditioned media from these cell types to analyze their stimulatory potentials.

Methods: Isolated progenitors were seeded within fibrinogen/thrombin gel-based constructs with or without supplementation with recombinant growth/differentiation factor-5 (GDF5). Early and late in culture, gene expression of differentiation markers and matrix assembly genes was analyzed. Tendon construct ultrastructure was also compared after 45 days. Moreover, conditioned media from tendon proper-derived progenitors, peritenon-derived progenitors, or tenocytes was applied to each of the three cell types to determine paracrine stimulatory effects of the factors secreted from each of the respective cell types.

Results: The cell orientation, extracellular domain and fibril organization of constructs were comparable to embryonic tendon. The tendon proper-derived progenitors produced a more tendon-like construct than the peritenon-derived progenitors. Seeded tendon proper-derived progenitors expressed greater levels of tenogenic markers and matrix assembly genes, relative to peritenon-derived progenitors. However, GDF5 supplementation improved expression of matrix assembly genes in peritenon progenitors and structurally led to increased mean fibril diameters. It also was found that peritenon-derived progenitors secrete factor(s) stimulatory to tenocytes and tendon proper progenitors.

Conclusions: Data demonstrate that, relative to peritenon-derived progenitors, tendon proper progenitors have greater potential for forming functional tendon-like tissue. Furthermore, factors secreted by peritenon-derived progenitors suggest a trophic role for this cell type as well. Thus, these findings highlight the synergistic potential of including these progenitor populations in restorative tendon engineering strategies.

No MeSH data available.


Fibrillar collagens were present in the tendon constructs. Staining for collagens I, III and V was analyzed in P1 Achilles tendon and progenitor-derived tendon constructs at Day 7. Because the tissue and cells originate from ScxGFP transgenic mice, cells with an active Scx promoter contain green fluorescent protein (GFP). In the P1 Achilles tendon, GFP allows for delineation (white dashed line) of peritenon (peri) and tendon proper (tp) (A-D). Active GFP was also detected for TP-derived progenitors in constructs (white arrows, E-L). As with the P1 Achilles tendon, TP, TP + GDF5, PERI, and PERI + GDF5 constructs were positive for staining for collagen I (A, E, I, M, Q), collagen III (B, F, J, N, R) and collagen V (C, G, K, O, S). Negative controls represent staining with secondary antibody only (D, H, L, P, T) (Bar: 100 μm).
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Figure 6: Fibrillar collagens were present in the tendon constructs. Staining for collagens I, III and V was analyzed in P1 Achilles tendon and progenitor-derived tendon constructs at Day 7. Because the tissue and cells originate from ScxGFP transgenic mice, cells with an active Scx promoter contain green fluorescent protein (GFP). In the P1 Achilles tendon, GFP allows for delineation (white dashed line) of peritenon (peri) and tendon proper (tp) (A-D). Active GFP was also detected for TP-derived progenitors in constructs (white arrows, E-L). As with the P1 Achilles tendon, TP, TP + GDF5, PERI, and PERI + GDF5 constructs were positive for staining for collagen I (A, E, I, M, Q), collagen III (B, F, J, N, R) and collagen V (C, G, K, O, S). Negative controls represent staining with secondary antibody only (D, H, L, P, T) (Bar: 100 μm).

Mentions: To analyze levels of differentiation markers and matrix assembly proteins within the constructs, expression of scleraxis, collagen I, collagen III, and collagen V in early (Day 7) constructs was evaluated using immuno-histochemical analyses. Scleraxis expression was visualized via the Scx promoter-driven green fluorescent protein (GFP) reporter present within cells derived from the ScxGFP mouse line [25]. GFP was detected in all tenocytes of P1 Achilles tendon yet not in cells of the surrounding peritenon (Figure 6A-D) Moreover, fewer cells expressed GFP in TP progenitor-derived constructs (Figure 6E-L); however, GFP was virtually absent in peritenon progenitor-derived onstructs (Figure 6M-T), even when supplemented with GDF5. Expression of collagen I was detectable in Achilles tendon (Figure 6A) as well as the tendon constructs (Figure 6E,I,M,Q). Collagen III was detectable in Achilles tendon (Figure 6B) and the tendon constructs (Figure 6F,J,N,R). Likewise, collagen V was present in the P1 Achilles tendon (Figure 6C) and the tendon constructs (Figure 6G,K,O,S). Thus, expression of fibrillar collagens is confirmed in the tendon constructs, and differential expression of the tendon marker scleraxis among progenitor-derived constructs is corroborated by Scx promoter-driven GFP.


Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

Mienaltowski MJ, Adams SM, Birk DE - Stem Cell Res Ther (2014)

Fibrillar collagens were present in the tendon constructs. Staining for collagens I, III and V was analyzed in P1 Achilles tendon and progenitor-derived tendon constructs at Day 7. Because the tissue and cells originate from ScxGFP transgenic mice, cells with an active Scx promoter contain green fluorescent protein (GFP). In the P1 Achilles tendon, GFP allows for delineation (white dashed line) of peritenon (peri) and tendon proper (tp) (A-D). Active GFP was also detected for TP-derived progenitors in constructs (white arrows, E-L). As with the P1 Achilles tendon, TP, TP + GDF5, PERI, and PERI + GDF5 constructs were positive for staining for collagen I (A, E, I, M, Q), collagen III (B, F, J, N, R) and collagen V (C, G, K, O, S). Negative controls represent staining with secondary antibody only (D, H, L, P, T) (Bar: 100 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230637&req=5

Figure 6: Fibrillar collagens were present in the tendon constructs. Staining for collagens I, III and V was analyzed in P1 Achilles tendon and progenitor-derived tendon constructs at Day 7. Because the tissue and cells originate from ScxGFP transgenic mice, cells with an active Scx promoter contain green fluorescent protein (GFP). In the P1 Achilles tendon, GFP allows for delineation (white dashed line) of peritenon (peri) and tendon proper (tp) (A-D). Active GFP was also detected for TP-derived progenitors in constructs (white arrows, E-L). As with the P1 Achilles tendon, TP, TP + GDF5, PERI, and PERI + GDF5 constructs were positive for staining for collagen I (A, E, I, M, Q), collagen III (B, F, J, N, R) and collagen V (C, G, K, O, S). Negative controls represent staining with secondary antibody only (D, H, L, P, T) (Bar: 100 μm).
Mentions: To analyze levels of differentiation markers and matrix assembly proteins within the constructs, expression of scleraxis, collagen I, collagen III, and collagen V in early (Day 7) constructs was evaluated using immuno-histochemical analyses. Scleraxis expression was visualized via the Scx promoter-driven green fluorescent protein (GFP) reporter present within cells derived from the ScxGFP mouse line [25]. GFP was detected in all tenocytes of P1 Achilles tendon yet not in cells of the surrounding peritenon (Figure 6A-D) Moreover, fewer cells expressed GFP in TP progenitor-derived constructs (Figure 6E-L); however, GFP was virtually absent in peritenon progenitor-derived onstructs (Figure 6M-T), even when supplemented with GDF5. Expression of collagen I was detectable in Achilles tendon (Figure 6A) as well as the tendon constructs (Figure 6E,I,M,Q). Collagen III was detectable in Achilles tendon (Figure 6B) and the tendon constructs (Figure 6F,J,N,R). Likewise, collagen V was present in the P1 Achilles tendon (Figure 6C) and the tendon constructs (Figure 6G,K,O,S). Thus, expression of fibrillar collagens is confirmed in the tendon constructs, and differential expression of the tendon marker scleraxis among progenitor-derived constructs is corroborated by Scx promoter-driven GFP.

Bottom Line: Tendon construct ultrastructure was also compared after 45 days.It also was found that peritenon-derived progenitors secrete factor(s) stimulatory to tenocytes and tendon proper progenitors.Data demonstrate that, relative to peritenon-derived progenitors, tendon proper progenitors have greater potential for forming functional tendon-like tissue.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Multipotent progenitor populations exist within the tendon proper and peritenon of the Achilles tendon. Progenitor populations derived from the tendon proper and peritenon are enriched with distinct cell types that are distinguished by expression of markers of tendon and vascular or pericyte origins, respectively. The objective of this study was to discern the unique tenogenic properties of tendon proper- and peritenon-derived progenitors within an in vitro model. We hypothesized that progenitors from each region contribute differently to tendon formation; thus, when incorporated into a regenerative model, progenitors from each region will respond uniquely. Moreover, we hypothesized that cell populations like progenitors were capable of stimulating tenogenic differentiation, so we generated conditioned media from these cell types to analyze their stimulatory potentials.

Methods: Isolated progenitors were seeded within fibrinogen/thrombin gel-based constructs with or without supplementation with recombinant growth/differentiation factor-5 (GDF5). Early and late in culture, gene expression of differentiation markers and matrix assembly genes was analyzed. Tendon construct ultrastructure was also compared after 45 days. Moreover, conditioned media from tendon proper-derived progenitors, peritenon-derived progenitors, or tenocytes was applied to each of the three cell types to determine paracrine stimulatory effects of the factors secreted from each of the respective cell types.

Results: The cell orientation, extracellular domain and fibril organization of constructs were comparable to embryonic tendon. The tendon proper-derived progenitors produced a more tendon-like construct than the peritenon-derived progenitors. Seeded tendon proper-derived progenitors expressed greater levels of tenogenic markers and matrix assembly genes, relative to peritenon-derived progenitors. However, GDF5 supplementation improved expression of matrix assembly genes in peritenon progenitors and structurally led to increased mean fibril diameters. It also was found that peritenon-derived progenitors secrete factor(s) stimulatory to tenocytes and tendon proper progenitors.

Conclusions: Data demonstrate that, relative to peritenon-derived progenitors, tendon proper progenitors have greater potential for forming functional tendon-like tissue. Furthermore, factors secreted by peritenon-derived progenitors suggest a trophic role for this cell type as well. Thus, these findings highlight the synergistic potential of including these progenitor populations in restorative tendon engineering strategies.

No MeSH data available.