Conventional allograft therapy for corneal scarring is widespread and successful, but donor tissue is not universally available, and some grafts fail owing to rejection and complications such as endothelial failure. We investigated direct treatment of corneal scarring using autologous stem cells, a therapy that, if successful, could reduce the need for corneal grafts. Mesenchymal cells were expanded from small superficial, clinically replicable limbal biopsies of human cadaveric corneo-scleral rims. Limbal biopsy–derived stromal cells (LBSCs) expanded rapidly in media containing human serum, were highly clonogenic, and could generate spheres expressing stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). Human LBSCs differentiated into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS) and organized a thick lamellar stroma-like tissue containing aligned collagen and keratan sulfate proteoglycans when cultured on aligned nanofiber substrata. When engrafted into mouse corneal wounds, LBSCs prevented formation of light-scattering scar tissue containing fibrotic matrix components. The presence of LBSCs induced regeneration of ablated stroma with tissue exhibiting lamellar structure and collagen organization indistinguishable from that of native tissue. Because the limbus can be easily biopsied from either eye of an affected individual and LBSCs capable of corneal stromal remodeling can be expanded under xeno-free autologous conditions, these cells present a potential for autologous stem cell–based treatment of corneal stromal blindness.

The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes.

The small leucine-rich proteoglycans (SLRPs), decorin and biglycan, are key regulators of collagen fibril and matrix assembly. The goal of this work was to elucidate the roles of decorin and biglycan in tendon homeostasis. Our central hypothesis is that decorin and biglycan expression in the mature tendon would be critical for the maintenance of the structural and mechanical properties of healthy tendons. Defining the function(s) of these SLRPs in tendon homeostasis requires that effects in the mature tendon be isolated from their influence on development. Thus, we generated an inducible knockout mouse model that permits genetic ablation of decorin and biglycan expression in the mature tendon, while maintaining normal expression during development. Decorin and biglycan expression were knocked out in the mature patellar tendon with the subsequent turnover of endogenous SLRPs deposited prior to induction. The acute absence of SLRP expression was associated with changes in fibril structure with a general shift to larger diameter fibrils in the compound knockout tendons, together with fibril diameter heterogeneity. In addition, tendon mechanical properties were altered. Compared to wild-type controls, acute ablation of both genes resulted in failure of the tendon at lower loads, decreased stiffness, a trend toward decreased dynamic modulus, as well as a significant increase in percent relaxation and tissue viscosity. Collagen fiber realignment was also increased with a delayed and slower in response to load in the absence of expression. These structural and functional changes in response to an acute loss of decorin and biglycan expression in the mature tendon demonstrate a significant role for these SLRPs in adult tendon homeostasis.

Specific niches may affect how cells from different regions contribute to tendon biology, particularly in regard to the healing of certain tendinopathies. The objectives of this study are to determine whether distinct subpopulations of stem/progenitor cells are found within the tendon proper and the epi- and paratenon, the peritenon, as well as to characterize these stem/progenitor cell populations. In this study, we hypothesized that tendon stem/progenitor cells exist in each region, that these populations possess distinct features, and that these populations while multipotent could have differing potentials. To test this hypothesis, stem/progenitor cells were isolated and characterized from the peritenon and tendon proper of mouse Achilles tendons. Colony-forming unit and multipotency assays, as well as flow cytometry, and real-time quantitative polymerase chain reaction analyses of stem cell markers were performed. Significantly, more stem/progenitor cell colonies were observed from cells derived from the tendon proper relative to the peritenon. Analysis of surface markers for stem/progenitor cells from both regions indicated that they were Sca1(+) (stem cell marker), Cd90(+) and Cd44(+) (fibroblast markers), Cd18(-) (leukocyte marker), Cd34(-) (hematopoietic and vascular marker), and Cd133(-) (perivascular marker). Tendon proper stem/progenitor cells had increased expression levels for tenomodulin (Tnmd) and scleraxis (Scx), indicative of enrichment of stem/progenitor cells of a tendon origin. In contrast, cells of the peritenon demonstrated relative increases in the vascular (endomucin) and pericyte (Cd133) markers relative to cells from the tendon proper. Stem/progenitor cells from both regions were multipotent (adipogenic, chondrogenic, osteogenic, and tenogenic). These findings demonstrated that different progenitor populations exist within discrete niches of the Achilles tendon-tendon proper versus peritenon. Overall, these data support the hypothesis that the progenitor pools from both regions have distinct properties and contain enriched progenitor subpopulations of different origins. Moreover, in considering their roles in tendon healing more broadly, they are potential cell sources that may differentially contribute to intrinsic and extrinsic tendon repair mechanisms. That is, intrinsic repair may require a progenitor class with predominant tendon marker expression, while extrinsic repair may involve a progenitor class recruited from perivascular cells of the peritenon.

SummaryCollagen V is a regulatory fibril-forming collagen that forms heterotypic fibrils with collagen I. Deletion of collagen V in the mouse is associated with a lack of fibril assembly in the embryonic mesenchyme, with a resultant lethal phenotype. The current work elucidates the regulatory roles of collagen V during development and growth of tissues. A conditional mouse model with a mutation in Col5a1 was developed using a Cre-loxP approach. Col5a1 was ablated in Col5a1 flox/flox mice using a cornea stroma-specific Kera-Cre driver mouse to produce a bitransgenic Col5a1Dst/Dst line that is null for collagen V. This permits analyses of the corneal stroma, a widely used model for studies of collagen V. The collagen-V-knockout stroma demonstrated severe dysfunctional regulation of fibrillogenesis. Fibril diameters were significantly increased, with an abnormal, heterogeneous distribution; fibril structure was abnormal, fibril number was decreased and lamellae were disorganized with decreased stroma thickness. The phenotype was more severe in the anterior versus posterior stroma. Opacity was demonstrated throughout the Col5a1Dst/Dst stroma, with significantly increased haze intensity compared with control mice. These data indicate central regulatory roles for collagen V in fibril and matrix assembly during tissue development, with dysfunctional regulation resulting in a functional loss of transparency.