Regeneration of collagen fibrils at the papillary dermis by reconstructing basement membrane at the dermal-epidermal junction.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Acellular and cellular high-density, collagen-fibril constructs with suprafibrillar organization.

Collagen is used extensively for tissue engineering due to its prevalence in connective tissues and its role in defining tissue biophysical and biological signalling properties. However, traditional collagen-based materials fashioned from atelocollagen and telocollagen have lacked collagen densities, multi-scale organization, mechanical integrity, and proteolytic resistance found within tissues in vivo. Here, highly interconnected low-density matrices of D-banded fibrils were created from collagen oligomers, which exhibit fibrillar as well as suprafibrillar assembly. Confined compression then was applied to controllably reduce the interstitial fluid while maintaining fibril integrity. More specifically, low-density (3.5 mg mL(-1)) oligomer matrices were densified to create collagen-fibril constructs with average concentrations of 12.25 mg mL(-1) and 24.5 mg mL(-1). Control and densified constructs exhibited nearly linear increases in ultimate stress, Young's modulus, and compressive modulus over the ranges of 65 to 213 kPa, 400 to 1.26 MPa, and 20 to 150 kPa, respectively. Densification also increased construct resistance to collagenase degradability. Finally, this process was amenable to creating high-density cellularized tissues; all constructs maintained high cell viability (at least 97%) immediately following compression as well as after 1 day and 7 days of culture. This method, which integrates the suprafibrillar assembly capacity of oligomers and controlled fluid reduction by confined compression, supports the rational and scalable design of a broad range of collagen-fibril materials and cell-encapsulated tissue constructs for tissue engineering applications.

Fibrillar, fibril-associated and basement membrane collagens of the arterial wall: architecture, elasticity and remodeling under stress.

The ability of a human artery to pass through 150 million liters of blood sustaining 2 billion pulsations of blood pressure with minor deterioration depends on unique construction of the arterial wall. Viscoelastic properties of this construction enable to re-seal the occuring damages apparently without direct immediate participance of the constituent cells. Collagen structures are considered to be the elements that determine the mechanoelastic properties of the wall in parallel with elastin responsible for elasticity and resilience. Collagen scaffold architecture is the function-dependent dynamic arrangement of a dozen different collagen types composing three distinct interacting forms inside the extracellular matrix of the wall. Tightly packed molecules of collagen types I, III, V provide high tensile strength along collagen fibrils but toughness of the collagen scaffold as a whole depends on molecular bonds between distinct fibrils. Apart of other macromolecules in the extracellular matrix (ECM), collagen-specific interlinks involve microfilaments of collagen type VI, meshwork-organized collagen type VIII, and FACIT collagen type XIV. Basement membrane collagen types IV, XV, XVIII and cell-associated collagen XIII enable transmission of mechanical signals between cells and whole artery matrix. Collagen scaffold undergoes continuous remodeling by decomposition promoted with MMPs and reconstitution from newly produced collagen molecules. Pulsatile stress-strain load modulates both collagen synthesis and MMP-dependent collagen degradation. In this way the ECM structure becomes adoptive to mechanical challenges. The mechanoelastic properties of the arterial wall are changed in atherosclerosis concomitantly with collagen turnover both type-specific and dependent on the structure. Improving the feedback could be another approach to restore sufficient blood circulation.

Secreted protein acidic and rich in cysteine (SPARC)-null mice exhibit more uniform outflow.

PURPOSE: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) in many tissues and is highly expressed in trabecular meshwork (TM). SPARC-null mice have a 15% to 20% decrease in intraocular pressure (IOP) compared to wild-type (WT) mice. We hypothesized that mouse aqueous outflow is segmental, and that transgenic deletion of SPARC causes a more uniform pattern that correlates with IOP and TM morphology. METHODS: Eyes of C57BL6-SV129 WT and SPARC-null mice were injected with fluorescent microbeads, which were also passively exposed to freshly enucleated eyes. Confocal and electron microscopy were performed. Percentage effective filtration length (PEFL) was calculated as PEFL = FL/TL × 100%, where TL = total length and FL = filtration length. IOP was measured by rebound tonometry. RESULTS: Passive microbead affinity for WT and SPARC-null ECM did not differ. Segmental flow was observed in the mouse eye. SPARC-null mice had a 23% decrease in IOP. PEFL increased in SPARC-null (70.61 ± 11.36%) versus WT mice (54.68 ± 9.95%, P < 0.005; n = 11 pairs), and PEFL and IOP were negatively correlated (R(2) = 0.72, n = 10 pairs). Morphologically, TM of high-tracer regions had increased separation between beams compared to low-tracer regions. Collagen fibril diameter decreased in SPARC-null (28.272 nm) versus WT tissue (34.961 nm, P < 0.0005; n = 3 pairs). CONCLUSIONS: Aqueous outflow in mice is segmental. SPARC-null mice demonstrated a more uniform outflow pattern and decreased collagen fibril diameter. Areas of high flow had less compact juxtacanalicular connective tissue ECM, and IOP was inversely correlated with PEFL. Our data show a correlation between morphology, aqueous outflow, and IOP, indicating a modulatory role of SPARC in IOP regulation.

[Functional histology of dermis]. / Histologie fonctionnelle du derme.

The skin is composed of epidermis, dermis and subcutaneous tissue that interconnect anatomically. The dermis is an integrated system of fibrous and amorphous connective tissue that accommodates nerve and vascular networks, epidermally derived appendages, fibroblasts, macrophages and mast cells. Elastic and collagen tissue are the main types of fibrous connective tissue. The elastic connective tissue is assembled in a continuous network including mature elastic fibers, immature elaunin fibers and oxytalan fibers. Mature elastic fibers and elaunin have microfibrillar and amorphous matrix components while oxytalan fibers only contain microfibrils. Several molecules have been identified as constituents of the elastic fibers. Among the most characterized of these molecules is elastin in amorphous matrix, fibrillins 1 and 2 and LTBP-2 (ligand of latent TGFbeta) in microfibrils and fibulins which interconnect elastin and fibrillins. Elastic fibers provides elasticity to the skin. Under electron microscope, collagen fibers appears as of bundles of periodically banded fibrils which are composed of collagens types I, III and V; type V collagen is believed to assist in regulating fibril diameter. They are associated with FACITs (fibril-associated collagen with interrupted triple helixes) collagens types XIV et XVI. Collagen fibers provide tensile strength to the skin. Non fibrous connective tissue molecules include finely filamentous glycoproteins, glycosaminoglycans and proteoglycans of "the ground substance" (hyaluronic acid and chondroitin sulphate, dermatan sulphate, versican, decorin). Fibroblasts, macrophages and mast cells are regular residents of the dermis. The main function of these cells are well known. Fibroblasts are responsible for the synthesis and the degradation of fibrous and non fibrous connective tissue matrix proteins. Macrophages are phagocytic; they process and present antigen to immunocompetent lymphoid cells. Mast cells are responsible for IgE mediated acute, subacute and chronic inflammation. All these cells have a long list of other functions, in particular they are involved in coagulation, wound healing and tissue remodeling.

The role of collagen II and cartilage fibril-associated molecules in skeletal development.

OBJECTIVE: The extracellular matrix (ECM) of hyaline cartilage contains an elaborated collagen fibrillar network, which is essential for the mechanical stability and the proper function of the tissue. Cartilage collagen fibrils consist of collagen II, the quantitatively minor collagens IX and XI, and several non-collagenous fibril-associated proteins. To understand the role some of these molecules in skeletal development, we have generated transgenic mouse strains harboring ablated genes for collagens II and IX, and matrilin-1. DESIGN: Mice lacking collagen II, collagen IX and matrilin-1 have been established earlier in our laboratory using standard techniques. To determine the consequences of the null mutations we used skeletal staining, histochemical and immunohistochemical assays, in situ hybridization and ultrastructural analysis. RESULTS: Transgenic mice deficient in collagen II (Col2a1-/-) die at birth and display a severely malformed skeleton characterized by abnormal endochondral ossification and impaired intervertebral disc development. Mice lacking collagen IX (Col9a1-/-) are viable and develop an osteoarthritis-like phenotype in knee joints between 9-12 months of age. To test the possibility that the reduction in collagen II content has an influence on the onset of degenerative changes of articular cartilage, we have generated mice, which are heterozygous for the collagen II null mutation and homozygous for the collagen IX null mutation. Col2a1+/- Col9a1-/- mice show no accelerated development of osteoarthritis compared with the collagen IX knockout animals. Finally, mice lacking matrilin-1, a non-collagenous glycoprotein that binds to both collagen fibrils and aggrecan, develop normally without detectable abnormalities in their skeleton. CONCLUSIONS: Our transgenic mouse strains carrying null mutations in genes encoding cartilage ECM proteins demonstrate that these proteins have different roles during skeletal development. Collagen II is important for cartilage formation, collagen IX for cartilage maintenance and matrilin-1 is redundant.