Relations between in vitro cytotoxicity and crosslinked dermal sheep collagens.

Collagen-based biomaterials have found various applications in the biomedical field. However, collagen-based biomaterials may induce cytotoxic effects. This study evaluated possible cytotoxic effects of (crosslinked) dermal sheep collagen (DSC) using a 7-d-methylcellulose cell culture with human skin fibroblasts. Non-crosslinked DSC (NDSC), hexamethylene-diisocyanate-crosslinked DSC (HDSC), and glutaraldehyde-crosslinked DSC (GDSC), their extracts (1 x 10 d to 4 x 10 d extracts), or the corresponding extracted DSC samples were tested. Cell growth was evaluated by cell counting, while cell morphology was assessed by light microscopy and transmission-electron microscopy. Both GDSC and, to a lesser extent, HDSC, induced cytotoxicity, observed as inhibited cell growth and deviant cell morphology. The deviant morphology consisted of extensive accumulations of lipid, reduction in the amount and dilatation of rough endoplasmatic reticulum, increased inclusions of cell remnants, and relatively rounded cell membranes. With HDSC, both primary cytotoxicity, due to extractable products from the material, and secondary cytotoxicity, possibly due to a release of cytotoxic products resulting from enzymatic cell-biomaterial interactions, could be discriminated. With GDSC, however, no clear distinction between primary and secondary cytotoxicity could be made. With NDSC, only primary cytotoxicity, measured as low inhibition of cell proliferation, but without deviant morphology, was observed. These remarkable differences in cytotoxicity are discussed in relation to residual agents and specific crosslinks present in DSCs as a consequence of processing and the crosslinking agents used. The residual agents and the specific crosslinks give rise to differences in direct release of products and in sensitivity to hydrolysis and enzymatic breakdown.     

Injectable cross-linked collagen with improved flow properties

Aqueous suspensions of glutaraldehyde cross-linked fibrillar collagen and non-cross-linked fibrillar collagen were examined by rheometry, particle size analysis, and microscopic techniques. Although cross-linked collagen suspensions were similar to non-cross-linked suspensions by microscopic and size analyses, they differed in rheometric properties. Concentric cylinder Couette flow, shear creep, uniaxial creep, and porous bed flow all revealed that cross-linked collagen was more resistant to deformation and flow than non-cross-linked collagen. These results were in agreement with in vivo dermal implantation studies, both in pig and human; i.e., compared to non-cross-linked collagen, the cross-linked formulation was more difficult to inject into tissue and did not spread uniformly, sometimes giving rise to palpable lumps or large masses evident in histological sections. When hyaluronic acid was blended with cross-linked collagen to achieve a final hyaluronate concentration of 5 mg/mL, there was a significant improvement in ease of injection into tissue. Rheometry on blends of hyaluronate and cross-linked collagen demonstrated that the blend required lower forces to achieve deformation and flow, compared to cross-linked collagen alone. Particle size analysis on the blend showed a reduction in fiber aggregate dimensions, compared to cross-linked collagen alone.     

An injectable cross-linked scaffold for nucleus pulposus regeneration

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite collagen-hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated intervertebral discs.     

3H-collagen turnover in non-cross-linked and aldehyde-cross-linked dermal collagen grafts.

Using trypsin-purified rat dermal collagen labelled with tritiated hydroxyproline and proline, a study has been made of hydroxyproline turnover in non-cross-linked and glutaraldehyde- and formaldehyde-cross-linked collagen when implanted s.c. in unlabelled isogenic rats. Grafts cross-linked with 0.01% glutaraldehyde maintained their collagen mass over a 22-week period, loss of original collagen being balanced by the gain in new collagen (22% at 22 weeks). Cross-linking with 5% formaldehyde temporarily inhibited collagen loss as compared with non-cross-linked grafts. However, at 22 weeks both had lost some 30% of their collagen mass, the gain of new collagen (some 8%) only partially compensating for the loss of original implant collagen.     

3H-collagen turnover in non-cross-linked and aldehyde-cross-linked dermal collagen grafts

Using trypsin-purified rat dermal collagen labelled with tritiated hydroxyproline and proline, a study has been made of hydroxyproline turnover in non-cross-linked and glutaraldehyde- and formaldehyde-cross-linked collagen when implanted s.c. in unlabelled isogenic rats. Grafts cross-linked with 0.01% glutaraldehyde maintained their collagen mass over a 22-week period, loss of original collagen being balanced by the gain in new collagen (22% at 22 weeks). Cross-linking with 5% formaldehyde temporarily inhibited collagen loss as compared with non-cross-linked grafts. However, at 22 weeks both had lost some 30% of their collagen mass, the gain of new collagen (some 8%) only partially compensating for the loss of original implant collagen.     

An in vitro cytotoxicity study of aldehyde-treated pig dermal collagen.

The cytotoxicity of aldehyde-treated collagen was assayed by measuring 3H-thymidine incorporation in adult human skin fibroblasts grown in tissue culture for 1 or 3 days in the presence of pig dermal collagen cross-linked with formaldehyde or glutaraldehyde. A comparison was also made with collagen preparations washed for 2 weeks either at 15 degrees throughout or partly at 15 degrees and partly at 37 degrees. Collagen treated with both formaldehyde and glutaraldehyde proved increasingly toxic with increase in the concentrations of aldehyde used. While the maximum toxic effect was observed after 1 day culture in formaldehyde-treated collagen, with thymidine uptake ranging from 4-48% of control values with 5-0.1% formaldehyde and a 15 degrees wash, the toxic effect of glutaraldehyde treatment increased with longer exposure and at 3 days thymidine uptake ranged from 3-40% of control values with 0.05-0.001% glutaraldehyde and washing at 15 degrees. Washing partly at 37 degrees significantly reduced toxicity, the differences in thymidine uptake as compared with washing at 15 degrees alone ranging from 34-50% with 1 and 0.3% formaldehyde respectively in 1 day cultures and from 14-37% with 0.02 and 0.005% glutaraldehyde in 3 day cultures. While fibroblasts actively grew and migrated when seeded on non-cross-linked collagen, only limited cell survival occurred on aldehyde-treated collagen.     

An in vitro cytotoxicity study of aldehyde-treated pig dermal collagen

The cytotoxicity of aldehyde-treated collagen was assayed by measuring 3H-thymidine incorporation in adult human skin fibroblasts grown in tissue culture for 1 or 3 days in the presence of pig dermal collagen cross-linked with formaldehyde or glutaraldehyde. A comparison was also made with collagen preparations washed for 2 weeks either at 15 degrees throughout or partly at 15 degrees and partly at 37 degrees. Collagen treated with both formaldehyde and glutaraldehyde proved increasingly toxic with increase in the concentrations of aldehyde used. While the maximum toxic effect was observed after 1 day culture in formaldehyde-treated collagen, with thymidine uptake ranging from 4-48% of control values with 5-0.1% formaldehyde and a 15 degrees wash, the toxic effect of glutaraldehyde treatment increased with longer exposure and at 3 days thymidine uptake ranged from 3-40% of control values with 0.05-0.001% glutaraldehyde and washing at 15 degrees. Washing partly at 37 degrees significantly reduced toxicity, the differences in thymidine uptake as compared with washing at 15 degrees alone ranging from 34-50% with 1 and 0.3% formaldehyde respectively in 1 day cultures and from 14-37% with 0.02 and 0.005% glutaraldehyde in 3 day cultures. While fibroblasts actively grew and migrated when seeded on non-cross-linked collagen, only limited cell survival occurred on aldehyde-treated collagen.     

In vivo degradation of processed dermal sheep collagen evaluated with transmission electron microscopy.

The in vivo degradation of hexamethylenediisocyanate-tanned dermal sheep collagen was studied with transmission electron microscopy. Discs of hexamethylenediisocyanate-tanned dermal sheep collagen were subcutaneously implanted in rats. Both an intra- and an extracellular route of degradation could be distinguished. In addition to normal components of a typical foreign body reaction, remarkable phenomena, such as locally deviant neutrophil morphology, infiltration of basophil-like cells, indications of foreign body multinucleate giant cells formed from different cell types, aluminium silicate accumulations and calcium phosphate depositions, were observed. Foreign body multinucleate giant cells intracellularly degraded hexamethylenediisocyanate-tanned dermal sheep collagen after internalization. Both internalized and cellularly enveloped hexamethylenediisocyanate-tanned dermal sheep collagen degraded by the detachment of fibrils. Another extracellular route of degradation was characterized by calcium phosphate depositions in large bundles of hexamethylenediisocyanate-tanned dermal sheep collagen. From 6 wk, the hexamethylenediisocyanate-tanned dermal sheep collagen implant was replaced by rat connective tissue, which was subsequently also degraded. After 15 wk, the presence of basophil-like foreign body multinucleated giant cells containing aluminium/silicon-crystalline accumulations still persisted. These phenomena were related to the specific nature of the material used and suggest cytotoxicity. They emphasize the need for detailed evaluation at the ultrastructural level of newly developed biomaterials before they can be used for medical applications.     

Effect of aldehyde cross-linking on human dermal collagen implants in the rat.

The fate of s.c. implants of fibrous, trypsin-purified human dermal collagen and collagen cross-linked with formaldehyde and glutaraldehyde has been studied in rats. Dermal collagen, and untreated skin implants, underwent resorption associated with a pronounced round-cell reaction. While collagen implants cross-linked with solutions of 0.04% and 0.08% formaldehyde became reduced in size, those cross-linked with 1% formaldehyde and 0.01%, 0.02% and 0.04% glutaraldehyde, although undergoing some collagen remodelling, retained their original size over the 25-week period of study. At 5 weeks the aldehyde cross-linked implants showed their greatest cellularity, reaching a lower, more stable cell population by 18 weeks. More round cells were seen at 5 weeks, particularly after formaldehyde cross-linking, than a later times when few were present. The results indicate that aldehyde-stabilized preparations of heterograft dermal collagen could have applications in the repair of tissue defects in man.     

Effect of aldehyde cross-linking on human dermal collagen implants in the rat

The fate of s.c. implants of fibrous, trypsin-purified human dermal collagen and collagen cross-linked with formaldehyde and glutaraldehyde has been studied in rats. Dermal collagen, and untreated skin implants, underwent resorption associated with a pronounced round-cell reaction. While collagen implants cross-linked with solutions of 0.04% and 0.08% formaldehyde became reduced in size, those cross-linked with 1% formaldehyde and 0.01%, 0.02% and 0.04% glutaraldehyde, although undergoing some collagen remodelling, retained their original size over the 25-week period of study. At 5 weeks the aldehyde cross-linked implants showed their greatest cellularity, reaching a lower, more stable cell population by 18 weeks. More round cells were seen at 5 weeks, particularly after formaldehyde cross-linking, than a later times when few were present. The results indicate that aldehyde-stabilized preparations of heterograft dermal collagen could have applications in the repair of tissue defects in man.     

Biocompatible collagen scaffolds from a human amniotic membrane: physicochemical and in vitro culture characteristics

A reconstituted collagen membrane from human amnion has been investigated as a source of collagen matrix, which could be used as a substratum for culturing human fibroblasts. The suitability of pepsin-solubilized reconstituted human amniotic membrane, before and after cross-linking with chitosan, as a dermal matrix for culturing fibroblast was assessed by morphologic, physicochemical, cytotoxic and histochemical methods. Measurement of thermodynamic behaviour, by differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA), and tensile strength suggested that the cross-linked membrane had sufficient elasticity to serve as an efficient dermal substrate for in vitro culture of fibroblasts. Fibroblasts cultured on the chitosan cross-linked collagen membrane had good adherence, retaining their morphology as indicated by microscopic analysis. Proliferation of fibroblasts. observed on this membrane affirms its non-toxic nature. These results support the application of reconstituted human amniotic collagen membrane as collagenous scaffolds to culture fibroblasts in vitro.     

Biocompatible collagen scaffolds from a human amniotic membrane: physicochemical and in vitro culture characteristics

A reconstituted collagen membrane from human amnion has been investigated as a source of collagen matrix, which could be used as a substratum for culturing human fibroblasts. The suitability of pepsin-solubilized reconstituted human amniotic membrane, before and after cross-linking with chitosan, as a dermal matrix for culturing fibroblast was assessed by morphologic, physicochemical, cytotoxic and histochemical methods. Measurement of thermodynamic behaviour, by differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA), and tensile strength suggested that the cross-linked membrane had sufficient elasticity to serve as an efficient dermal substrate for in vitro culture of fibroblasts. Fibroblasts cultured on the chitosan cross-linked collagen membrane had good adherence, retaining their morphology as indicated by microscopic analysis. Proliferation of fibroblasts, observed on this membrane affirms its non-toxic nature. These results support the application of reconstituted human amniotic collagen membrane as collagenous scaffolds to culture fibroblasts in vitro.     

Retinal pigment epithelium cell alignment on nanostructured collagen matrices

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α(2) were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α(2) was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α(2) expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α(2)-mediated matrix binding was verified by preincubation with an α(2)-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.     

Physical,mechanical and degradation properties,and Schwann cell affinity of cross-linked chitosan films

Three kinds of cross-linked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as cross-linking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the cross-linked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with non-cross-linked chitosan films. The protein adsorption on chitosan films was determined by means of enzyme-linked immunosorbent assay (ELISA). In comparison with the non-cross-linked films, the chitosan films cross-linked with HDI showed a significant increase (up to 40–50%) in both fibronectin and laminin adsorption, while the protein adsorption on the other two kinds of cross-linked films was similar to that on non-cross-linked films. In addition, cell culture revealed that the HDI cross-linked chitosan films enhanced the spread and proliferation of Schwann cells while the other cross-linked films delayed the cell proliferation. These results suggest that HDI cross-linking of chitosan films provides a combination of physical properties, biodegradability and Schwann cell affinity suitable for peripheral nerve regeneration.     

The foreign body reaction to a biodegradable biomaterial differs between rats and mice

Before a biomaterial can be applied in the clinic, biocompatibility must be tested in in vivo models, by monitoring the foreign body reaction. In this study, we compared the foreign body reaction (FBR) to the biodegradable biomaterial hexamethylenediisocyanate crosslinked dermal sheep collagen (HDSC) between several strains of rats and mice. HDSC disks were implanted subcutaneously on the backs of AO, BN, F344, LEW, and PVG rats and on the backs of 129 SVEV, BALB/c, and C57BL/6 mice. Materials were explanted after 7, 14, 21, and 28 days and processed for (immuno) light and transmission electron microscopic evaluation. In all rat strains, giant cell formation and phagocytosis of HDSC bundles were comparable. In addition, in the PVG rat, many plasma cells infiltrated the HDSC disks. Only a few T cells were present in AO and PVG rats, whereas, in F344 and LEW rats, the presence of T cells was more pronounced. BN rats showed an intermediate T-cell infiltration. In mice, the FBR to HDSC was comparable between the different strains. Compared with rats, giant cell formation was limited, whereas stroma formation was more abundant. Phagocytosis of HDSC bundles rarely occurred in mice, whereas calcification was observed more often. It is concluded that the FBR to HDSC clearly differs between rats and mice. This has consequences for assessment studies on biocompatibility and also on fundamental biomaterial research.     

In vitro evaluation of a chitosan membrane cross-linked with genipin.

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties. antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

克隆化LAK细胞及其培养上清对白血病细胞的细胞毒和分化诱导作用

用半固体－液体两步法克隆的人ＬＡＫ细胞不仅表现对ＮＫ敏感的Ｋ５６２细胞和ＮＫ抵抗的ＨＬ－６０细胞强烈的细胞毒性，在二次杀瘤过程中表现同样高的活性，而且ＬＡＫ细胞培养上清对白血病细胞系ＨＬ－６０和新鲜白血病细胞具有增殖抑制和分化诱导作用。然而不同杀伤活性的ＬＡＫ细胞培养上清对新鲜白血病细胞增殖的影响不同。结果表明ＬＡＫ细胞在直接杀伤白血病细胞的同时，还通过分泌一些活性因子间接影响白血病细胞的增殖和促进其分化成熟，反映了ＬＡＫ细胞抗白血病作用的多向性。     

Human blood lymphocyte fractionation with special attention to their cytotoxic potential.

In studies concerning the natural cytotoxic activity of human blood lymphocytes we have found that: 1. T cell, which rosette with sheep erythrocytes can be separated by centrifuging on Ficoll the lymphocyte and erythrocyte mixture without previously pelleting and incubating in the cold. 2. Lysis of marker erythrocytes with ammonium chloride impairs the cytotoxic activity of lymphocytes. Incubation at 37 degrees C for 12 hr prior to the test restores the activity to some extent. 3. A high proportion of the so call "null" fraction i.e. cells remaining in the interface after removal of B cells (passage on nylon column) and sedimentation of E rosettes, sediments as rosettes with sheep EAC'indicating that these cells carry low density E and C3 receptors. Rosetting with SRBC or ox EAC' gave significantly lowr values. On a per cell basis the "null" fraction was the most efficient one in natural cytotoxicity. Depletion of cells with low affinity E or E and C3 receptors left highly active subfractions.     

Physical, mechanical and degradation properties, and schwann cell affinity of cross-linked chitosan films

Three kinds of cross-linked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as cross-linking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the cross-linked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with non-cross-linked chitosan films. The protein adsorption on chitosan films was determined by means of enzyme-linked immunosorbent assay (ELISA). In comparison with the non-cross-linked films, the chitosan films cross-linked with HDI showed a significant increase (up to 40-50%) in both fibronectin and laminin adsorption, while the protein adsorption on the other two kinds of cross-linked films was similar to that on non-crosslinked films. In addition, cell culture revealed that the HDI cross-linked chitosan films enhanced the spread and proliferation of Schwann cells while the other cross-linked films delayed the cell proliferation. These results suggest that HDI cross-linking of chitosan films provides a combination of physical properties, biodegradability and Schwann cell affinity suitable for peripheral nerve regeneration.     

Concanavalin A-mediated in Vitro Activation of a Secondary Cytotoxic T-Cell Response in Virus-primed Splenocytes

In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt to characterize further these effector cells and, in particular, to establish whether the Con A-activated cytotoxic effectors are qualitatively different from the secondary cytotoxic T cells induced by restimulation with the homologous antigen. It was found that: (1) in vitro activation with Con A could be obtained with populations harvested between 13 days (the earliest tested) and at least 300 days after priming; (2) cytotoxicity was independent of the presence of carried-over Con A in the cytotoxicity assay; (3) cytotoxicity was dependent on close association between activated T cells and target cells, since no evidence was found to indicate a role for other cell types or soluble (cytotoxic or arming) factors; (4) cytotoxicity was specific with regard to both virus and 'self'. By comparison with previous data on LCMV-induced cytotoxic T cells, it is concluded that Con A induces the generation of cytotoxic T cells from LCMV-primed splenocytes, which, by the criteria used, are indistinguishable from virus-induced secondary cytotoxic T cells. The implications of these findings are discussed.