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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.

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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.


Expression of tendon differentiation markers and matrix assembly genes is generally greater in tendon proper-derived progenitors. RT-qPCR analyses demonstrated greater expression of the tendon markers Scx(A) and Tnmd(B) in tendon proper (TP)-derived progenitors within their respective constructs. However, expression of the vascular marker Emcn(C) was greater in peritenon-derived (PERI) progenitors. Expression of Bgn(D) remains fairly constant across cell sources, while expression for Dcn(E) is greater in TP-derived progenitor-seeded tendon constructs. Expression of FACIT collagens Col12a1(F) and Col14a1(G) is greater early in culture, particularly for TP-derived progenitors. Gene expression for fibril-forming collagens Col1a1(H), Col3a1(I), and Col5a1(J) is greater in TP-derived progenitors. Expression of Col11a1(K) is greatest early in TP-derived progenitors. (Biological replicates, n = 5 to 8; Mann–Whitney-Wilcoxon test – P <0.01, *; P <0.05, #; fold changes and statistical significance further reported in Table 2).
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Figure 4: Expression of tendon differentiation markers and matrix assembly genes is generally greater in tendon proper-derived progenitors. RT-qPCR analyses demonstrated greater expression of the tendon markers Scx(A) and Tnmd(B) in tendon proper (TP)-derived progenitors within their respective constructs. However, expression of the vascular marker Emcn(C) was greater in peritenon-derived (PERI) progenitors. Expression of Bgn(D) remains fairly constant across cell sources, while expression for Dcn(E) is greater in TP-derived progenitor-seeded tendon constructs. Expression of FACIT collagens Col12a1(F) and Col14a1(G) is greater early in culture, particularly for TP-derived progenitors. Gene expression for fibril-forming collagens Col1a1(H), Col3a1(I), and Col5a1(J) is greater in TP-derived progenitors. Expression of Col11a1(K) is greatest early in TP-derived progenitors. (Biological replicates, n = 5 to 8; Mann–Whitney-Wilcoxon test – P <0.01, *; P <0.05, #; fold changes and statistical significance further reported in Table 2).

Mentions: To test our hypothesis that tendon proper- and peritenon-derived progenitors have different tenogenic capacities within the in vitro regenerative model, we analyzed expression of differentiation markers and matrix assembly genes early (Day 7) and late (Day 45) in construct formation (Table 2). Expression of tendon differentiation markers Scx and Tnmd was shown to be different in pre-seeded tendon proper-derived and peritenon-derived progenitors [4]; in this study, expression of Scx is greater in tendon proper-derived progenitors and trends higher for Tnmd (Figure 4A,B; Table 2). Expression of Scx and Tnmd was significantly greater in tendon proper-derived progenitors seeded in constructs (Figure 4A,B; TP versus PERI: 30- to 90-fold and 13- to 21-fold, respectively), while expression of vascular marker Emcn had a greater expression level in pre-seeded peritenon-derived progenitors and a greater trend of expression in peritenon-derived progenitors (Figure 4C; Table 2). Transcript levels for Bgn were relatively equivalent across cultures (group) and stage (days) (Figure 4D), but levels for Dcn were significantly greater in tendon proper-derived progenitors (Figure 4E). FACIT collagen genes Col12a1 and Col14a1 were significantly greater in tendon proper-derived progenitors at the early time point (Figure 4F,G). Expression of fibrillar collagen genes Col1a1, Col3a1, Col5a1, and Col11a1 were generally greater in tendon proper-derived progenitors seeded in constructs both early and late in culture (Figure 4H-K).


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

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

Expression of tendon differentiation markers and matrix assembly genes is generally greater in tendon proper-derived progenitors. RT-qPCR analyses demonstrated greater expression of the tendon markers Scx(A) and Tnmd(B) in tendon proper (TP)-derived progenitors within their respective constructs. However, expression of the vascular marker Emcn(C) was greater in peritenon-derived (PERI) progenitors. Expression of Bgn(D) remains fairly constant across cell sources, while expression for Dcn(E) is greater in TP-derived progenitor-seeded tendon constructs. Expression of FACIT collagens Col12a1(F) and Col14a1(G) is greater early in culture, particularly for TP-derived progenitors. Gene expression for fibril-forming collagens Col1a1(H), Col3a1(I), and Col5a1(J) is greater in TP-derived progenitors. Expression of Col11a1(K) is greatest early in TP-derived progenitors. (Biological replicates, n = 5 to 8; Mann–Whitney-Wilcoxon test – P <0.01, *; P <0.05, #; fold changes and statistical significance further reported in Table 2).
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Related In: Results  -  Collection

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Figure 4: Expression of tendon differentiation markers and matrix assembly genes is generally greater in tendon proper-derived progenitors. RT-qPCR analyses demonstrated greater expression of the tendon markers Scx(A) and Tnmd(B) in tendon proper (TP)-derived progenitors within their respective constructs. However, expression of the vascular marker Emcn(C) was greater in peritenon-derived (PERI) progenitors. Expression of Bgn(D) remains fairly constant across cell sources, while expression for Dcn(E) is greater in TP-derived progenitor-seeded tendon constructs. Expression of FACIT collagens Col12a1(F) and Col14a1(G) is greater early in culture, particularly for TP-derived progenitors. Gene expression for fibril-forming collagens Col1a1(H), Col3a1(I), and Col5a1(J) is greater in TP-derived progenitors. Expression of Col11a1(K) is greatest early in TP-derived progenitors. (Biological replicates, n = 5 to 8; Mann–Whitney-Wilcoxon test – P <0.01, *; P <0.05, #; fold changes and statistical significance further reported in Table 2).
Mentions: To test our hypothesis that tendon proper- and peritenon-derived progenitors have different tenogenic capacities within the in vitro regenerative model, we analyzed expression of differentiation markers and matrix assembly genes early (Day 7) and late (Day 45) in construct formation (Table 2). Expression of tendon differentiation markers Scx and Tnmd was shown to be different in pre-seeded tendon proper-derived and peritenon-derived progenitors [4]; in this study, expression of Scx is greater in tendon proper-derived progenitors and trends higher for Tnmd (Figure 4A,B; Table 2). Expression of Scx and Tnmd was significantly greater in tendon proper-derived progenitors seeded in constructs (Figure 4A,B; TP versus PERI: 30- to 90-fold and 13- to 21-fold, respectively), while expression of vascular marker Emcn had a greater expression level in pre-seeded peritenon-derived progenitors and a greater trend of expression in peritenon-derived progenitors (Figure 4C; Table 2). Transcript levels for Bgn were relatively equivalent across cultures (group) and stage (days) (Figure 4D), but levels for Dcn were significantly greater in tendon proper-derived progenitors (Figure 4E). FACIT collagen genes Col12a1 and Col14a1 were significantly greater in tendon proper-derived progenitors at the early time point (Figure 4F,G). Expression of fibrillar collagen genes Col1a1, Col3a1, Col5a1, and Col11a1 were generally greater in tendon proper-derived progenitors seeded in constructs both early and late in culture (Figure 4H-K).

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.