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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.


Related in: MedlinePlus

Progenitor-seeded fibrin gels form tendon-like structures. (A) Stem/progenitor cells are seeded within fibrinogen/thrombin; at Day 2, a distinct gel bridges the two silk sutures. (B) By Day 7, the fibrin within the structure has commenced contracting. (C) By Day 14, the construct has contracted into a long, narrow structure in which tension is applied uniaxially. Representative images are from tendon proper-derived stem/progenitor-cell-seeded constructs (Bar: 200 μm).
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Figure 1: Progenitor-seeded fibrin gels form tendon-like structures. (A) Stem/progenitor cells are seeded within fibrinogen/thrombin; at Day 2, a distinct gel bridges the two silk sutures. (B) By Day 7, the fibrin within the structure has commenced contracting. (C) By Day 14, the construct has contracted into a long, narrow structure in which tension is applied uniaxially. Representative images are from tendon proper-derived stem/progenitor-cell-seeded constructs (Bar: 200 μm).

Mentions: Tendon proper- and peritenon-derived progenitor cells were seeded into a regenerative tendon construct model as described previously [4,16]. Briefly, wells of six-well plates were coated with SYLGARD polymer (Dow Chemical, Midland, MI, USA). Within each well, two segments of size 0 silk were each pinned with a pair of minutiens insect pins (0.1 mm diameter, Fine Science Tools GmbH, Heidelberg, Germany) in each of the two suture segments positioned 10 mm apart. The contents of each well were sterilized by treatment with 100% ethanol, exposure to ultraviolet irradiation for 60 minutes and then rinsed in PBS. Within each well, 6.15 × 105 tendon proper or peritenon progenitor cells in 400 μl media, 83 μl of 20 mg/mL fibrinogen, and 10 μl of 200 U/mL thrombin (Sigma, St Louis, MO, USA) were combined and quickly spread over the polymer surface between the two suture segments. Plates were incubated at 37°C in alpha-MEM with 10% FBS, 100 U/ml penicillin, 100 μg/mL streptomycin, 250 ng/ml amphotericin B, 2 mM L-glutamine and 200 μM ascorbic-2-phosphate, with or without GDF5 (100 ng/ml r-mGDF5, R&D Systems, Minneapolis, MN, USA) [4,23,27]. Three times per week the plates were scored to release the fibrin gel as it contracted as previously described [16], and the culture medium was changed. Over the first two weeks, gels contracted and tendon constructs formed (Figure 1). Cultures were maintained for up to 45 days.


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

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

Progenitor-seeded fibrin gels form tendon-like structures. (A) Stem/progenitor cells are seeded within fibrinogen/thrombin; at Day 2, a distinct gel bridges the two silk sutures. (B) By Day 7, the fibrin within the structure has commenced contracting. (C) By Day 14, the construct has contracted into a long, narrow structure in which tension is applied uniaxially. Representative images are from tendon proper-derived stem/progenitor-cell-seeded constructs (Bar: 200 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Progenitor-seeded fibrin gels form tendon-like structures. (A) Stem/progenitor cells are seeded within fibrinogen/thrombin; at Day 2, a distinct gel bridges the two silk sutures. (B) By Day 7, the fibrin within the structure has commenced contracting. (C) By Day 14, the construct has contracted into a long, narrow structure in which tension is applied uniaxially. Representative images are from tendon proper-derived stem/progenitor-cell-seeded constructs (Bar: 200 μm).
Mentions: Tendon proper- and peritenon-derived progenitor cells were seeded into a regenerative tendon construct model as described previously [4,16]. Briefly, wells of six-well plates were coated with SYLGARD polymer (Dow Chemical, Midland, MI, USA). Within each well, two segments of size 0 silk were each pinned with a pair of minutiens insect pins (0.1 mm diameter, Fine Science Tools GmbH, Heidelberg, Germany) in each of the two suture segments positioned 10 mm apart. The contents of each well were sterilized by treatment with 100% ethanol, exposure to ultraviolet irradiation for 60 minutes and then rinsed in PBS. Within each well, 6.15 × 105 tendon proper or peritenon progenitor cells in 400 μl media, 83 μl of 20 mg/mL fibrinogen, and 10 μl of 200 U/mL thrombin (Sigma, St Louis, MO, USA) were combined and quickly spread over the polymer surface between the two suture segments. Plates were incubated at 37°C in alpha-MEM with 10% FBS, 100 U/ml penicillin, 100 μg/mL streptomycin, 250 ng/ml amphotericin B, 2 mM L-glutamine and 200 μM ascorbic-2-phosphate, with or without GDF5 (100 ng/ml r-mGDF5, R&D Systems, Minneapolis, MN, USA) [4,23,27]. Three times per week the plates were scored to release the fibrin gel as it contracted as previously described [16], and the culture medium was changed. Over the first two weeks, gels contracted and tendon constructs formed (Figure 1). Cultures were maintained for up to 45 days.

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.


Related in: MedlinePlus