Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces.

Bovine aortic endothelial cells were cultured on surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), in the presence of serum. Cells adhered, spread, proliferated, and reached confluency as observed on ungrafted tissue culture polystyrene dishes. A decrease in culture temperature released cells only from the grafted surfaces without enzymatic or ethylenediaminetetraacetic acid treatment. Upon lowering temperature, the culture surfaces changed from hydrophobic to hydrophilic owing to the hydration of grafted PIPAAm and thus weakened the cell attachment to the dishes. Released cells maintained cell-cell junctions composing monolayer cell sheets. Immunoblotting and immunofluorescence microscopy revealed that fibronectin (FN) was deposited and accumulated on the grafted surfaces during the culture. Furthermore, the deposited FN matrix adhering to cell sheets was also recovered from temperature-responsive surfaces by low-temperature treatment, while trypsin treatment destroyed the matrix. The recovery of FN by low-temperature treatment was as high as by physical scraping with a rubber blade. Temperature-responsive surfaces can provide a novel method to use cultured confluent cell sheets for tissue engineering, and also to elucidate structure and function of deposited extracellular matrix during cell culture.     

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.     

Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest

We have developed temperature-responsive cell culture surfaces to harvest intact cell sheets for tissue-engineering applications. Both cost and safety issues (e.g., prions, bovine spongiform encephalopathy) are compelling reasons to avoid use of animal-derived materials, including serum, in such culture. In the present study, synthetic cell-adhesive peptides are immobilized onto temperature-responsive polymer-grafted surfaces, and cell adhesion and detachment under serum-free conditions were examined. The temperature-responsive polymer poly(N-isopropylacrylamide) (PI-PAAm) was functionalized by copolymerization with a reactive comonomer having both a carboxyl group and an isopropylacrylamide group. These copolymers were covalently grafted onto tissue culture-grade polystyrene dishes. Synthetic cell-adhesive peptides were then immobilized onto these surfaces via carboxyl groups. Bovine aortic endothelial cells both adhered and spread on these surfaces even under serum-free conditions at 37 degrees C, similar to those in 10% serum-supplemented culture. Spread cells promptly detached from the surfaces on lowering culture temperatures below the lower critical solution temperature of the polymer, 32 degrees C. These surfaces would be useful for serumfree culture for tissue-engineering applications.     

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32 degrees C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37 degrees C, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20 degrees C without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets.     

Transplantable urothelial cell sheets harvested noninvasively from temperature-responsive culture surfaces by reducing temperature

Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured in vitro should enable us to avoid these complications. Transplantable urothelial cell sheets were obtained by utilizing a temperature-responsive cell culture method, and then examined by immunostaining and electron microscopy. Canine urothelium was produced on the surfaces of temperature-responsive culture dishes covalently bonded with the thermally sensitive polymer, poly(N-isopropylacrylamide). Stratified urothelial cell sheets were cultured and then harvested intact without enzymatic treatment from these dishes by reducing the temperature. Histological structure and cell-to-cell junctions were compared between these urothelial cell sheets and those harvested with dispase. All urothelial cell sheets were harvested from the bonded surfaces by reducing the culture temperature without the need for dispase. Electron microscopy revealed well-developed microridge, microvilli, and cell junction complexes. Conversely, these same cell features were destroyed by dispase treatment. Immunoblotting revealed that dispase fragmented occludin, whereas it remained unchanged in the intact urothelial cell sheets. Novel urothelial cell sheets obtained by culture on temperature-responsive culture surfaces were successfully harvested much less destructively than with dispase. This technology should prove useful in urinary tract tissue engineering in the near future.     

Temperature-responsive glass coverslips with an ultrathin poly(N-isopropylacrylamide) layer

A temperature-responsive cross-linked polymer gel was covalently grafted onto glass coverslips by electron beam irradiation. The grafted thickness and amount of polymer as well as the surface wettability increased with the initial monomer concentration. When the monomer concentration was 5 wt.%, the grafted polymer density was 0.84microgcm(-2), and cells adhered and spread on the surface at 37 degrees C, but detached at 20 degrees C. In contrast, when the monomer concentration was 35 wt.%, the polymer density was 1.28microgcm(-2), and the surfaces were cell repellent even at 37 degrees C. These results show a remarkable contrast to those obtained from temperature-responsive polymer-grafted tissue culture polystyrene dishes, since various types of cells showed temperature-dependent cell adhesion/detachment when the grafted density was around 2microgcm(-2) on these surfaces. We discuss the possible molecular mechanisms underlying this discrepancy.     

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.     

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.     

The use of biotin-avidin binding to facilitate biomodification of thermoresponsive culture surfaces

Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G_nRGDS (n=1, 6, 12, 16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G₁₂RGDS-immobilized surfaces in the absence of serum at 37 °C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 °C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.     

Reconstruction of functional tissues with cell sheet engineering

The field of tissue engineering has yielded several successes in early clinical trials of regenerative medicine using living cells seeded into biodegradable scaffolds. In contrast to methods that combine biomaterials with living cells, we have developed an approach that uses culture surfaces grafted with the temperature-responsive polymer poly(N-isoproplyacrylamide) that allows for controlled attachment and detachment of living cells via simple temperature changes. Using cultured cell sheets harvested from temperature-responsive surfaces, we have established cell sheet engineering to create functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, tracheal resection, and cardiac failure. Additionally, by exploiting the unique ability of cell sheets to generate three-dimensional tissues composed of only cultured cells and their deposited extracellular matrix, we have also developed methods to create thick vascularized tissues as well as, organ-like systems for the heart and liver. Cell sheet engineering therefore provides a novel alternative for regenerative medicine approaches that require the re-creation of functional tissue structures.     

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.     

Cell Attachment–Detachment Control on Temperature-Responsive Thin Surfaces for Novel Tissue Engineering

Temperature-responsive intelligent surfaces, prepared by the modification of an interface mainly with poly(N-isopropylacrylamide) and its derivatives, have been investigated. Such surfaces exhibit temperature-responsive hydrophilic/hydrophobic alterations with external temperature changes, which, in turn, result in thermally modulated attachment and detachment with cells. The advantage of this system is that cells cultured on such temperature-responsive surfaces can be recovered as single cells and/or confluent cell sheets, while keeping the deposited extracellular matrix intact, simply by lowering the temperature without conventional enzymatic treatment. Here, we focus and compare various methods of producing temperature-responsive surfaces for controlling cell attachment/detachment. Spontaneous cell attachment and detachment using several types of temperature-responsive surfaces are mentioned and various effects, such as film thickness and polymer conformation, are discussed. In addition, the development of the next generation of temperature-responsive surfaces using modifications of the polymer coating to allow for rapid cell recovery is summarized.     

Rapid cell sheet detachment from poly(N-isopropylacrylamide)-grafted porous cell culture membranes

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.     

Creation of myocardial tubes using cardiomyocyte sheets and an in vitro cell sheet-wrapping device

Regenerative medicine involving injection of isolated cells and transplantation of tissue-engineered myocardial patches, has received significant attention as an alternative method to repair damaged heart muscle. In the present study, as the next generation of myocardial tissue engineering we demonstrate the in vitro fabrication of pulsatile myocardial tubes using cell sheet engineering technologies. Three neonatal rat cardiomyocyte sheets, which were harvested from temperature-responsive culture dishes, were wrapped around fibrin tubes using a novel cell sheet-wrapping device. The tubular constructs demonstrated spontaneous, synchronized pulsation within 3h after cell sheet wrapping. Contractile force measurements showed that the contractile force increased in accordance with both increasing rest length (Starling mechanism) and increasing extracellular Ca(2+) concentration. Furthermore, the tissue-engineered myocardial tubes presented measurable inner pressure changes evoked by tube contraction (0.11+/-0.01mmHg, max 0.15mmHg, n=5). Histological analyses revealed both well-differentiated sarcomeres and diffuse gap junctions within the myocardial tissues that resembled native cardiac muscle. These data indicate that tissue-engineered myocardial tubes have native heart-like structure and function. These new myocardial tissue constructs should be useful for future applications in physiological studies and pharmacological screening, and present a possible core technology for the creation of engineered tissues capable of independent cardiac assistance.     

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.     

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.     

Cell sheet engineering: recreating tissues without biodegradable scaffolds

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.     

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.     

Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets

Recently researchers have attempted to bioengineer three-dimensional (3-D) myocardial tissues using cultured cells in order to repair damaged hearts. In contrast to the conventional approach of seeding cells onto 3-D biodegradable scaffolds, we have explored a novel technology called cell sheet engineering, which layers cell sheets to construct functional tissue grafts. In this study, in vivo survival, function, and morphology of myocardial tissue grafts were examined. Neonatal rat cardiomyocytes were noninvasively harvested as contiguous cell sheets from temperature-responsive culture dishes simply by reducing the culture temperature. Cardiomyocyte sheets were then layered and transplanted into the subcutaneous tissues of athymic rats. The microvasculature of the grafts was rapidly organized within a few days with macroscopic graft beatings observed 3 days after transplantation and preserved up to one year. Size, conduction velocity, and contractile force of transplanted grafts increased in proportion to the host growth. Histological studies showed characteristic structures of heart tissue, including elongated cardiomyocytes, well-differentiated sarcomeres, and gap junctions within the grafts. In conclusion, long-term survival and growth of pulsatile myocardial tissue grafts fabricated by layering cell sheets were confirmed, demonstrating that myocardial tissue regeneration based on cell sheet engineering may prove useful for permanent myocardial tissue repair.     

Comparison of proliferation and growth of human keratinocytes on plasma copolymers of acrylic acid/1,7-octadiene and self-assembled monolayers.

Human keratinocytes were cultured on plasma copolymers (PCPs), self-assembled monolayers (SAMs), and tissue culture poly(styrene) (TCPS). Plasma copolymerization was used to deposit films with controlled concentrations of carboxylic acid functional groups (<5%). Human keratinocytes were cultured onto these PCP surfaces, TCPS, and collagen I. A hydrocarbon plasma polymer surface was used as the negative control. Keratinocyte attachment was measured at 24 h and cell proliferation and growth at 3 and 7 days using optical microscopy and DNA concentrations. The PCP surfaces were compared with two SAM systems comprising pure acid and pure hydrocarbon functionalities, and pure gold was used as a control surface. PCP surfaces containing carboxylic acid functionalities promoted keratinocyte attachment. The level of attachment on these surfaces was comparable to that seen on collagen I, a preferred substratum for the culturing of keratinocytes. After several days in culture the cells were well attached and proliferative, forming confluent sheets of keratinocytes. This result was confirmed by DNA assays that suggested the acid PCP surfaces were performing as well as collagen I. Keratinocytes attached well to gold and acid-terminated SAMs but attached poorly to methyl-terminated SAMs. The acid functionality also promoted proliferation and growth of keratinocytes after several days in culture. DNA assays revealed that keratinocyte growth on the acid surface was higher than on collagen I.