Changes in mechanical properties and cellularity during long-term culture of collagen fiber ACL reconstruction scaffolds

Resorbable scaffolds for anterior cruciate ligament (ACL) reconstruction should provide temporary mechanical function then gradually breakdown while promoting matrix synthesis by local cells. Crosslinking influences collagen's mechanical properties, degradation rate, and interactions with cells. Our objective was to compare the effects of different crosslinkers on cellularity and mechanical properties during long-term (8 week) culture of collagen fiber scaffolds. Fibers were fabricated from an acid-insoluble dispersion of bovine dermal collagen and crosslinked with either ultraviolet irradiation (UV; a physical crosslinker) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; a chemical crosslinker). Scaffolds consisted of 50 fibers bundled in parallel. Initial attachment of fibroblasts was similar on both scaffolds; however, from 1 to 8 weeks in culture, UV-crosslinked scaffolds had significantly more cells attached than EDC-crosslinked scaffolds. The initial breaking load (3.50 N) and stiffness (2.23 N/mm) of EDC-crosslinked scaffolds were significantly greater than those of UV-crosslinked scaffolds (2.32 N; 1.21 N/mm) and were unaffected by long-term fibroblast culture. In contrast, the load-bearing capacity of fibroblast-seeded UV-crosslinked scaffolds decreased 60% to 0.91 N after 8 weeks in culture. EDC-crosslinked scaffolds maintained strength and moderate cellularity; UV-crosslinked scaffolds, in contrast, were highly cellular, but had poor mechanical properties that decreased during culture. These in vitro results suggest that collagen fiber scaffolds crosslinked with EDC may be more suitable for ACL reconstruction than those crosslinked with UV.     

Physical characterization of vascular grafts cultured in a bioreactor

Tubular scaffolds of collagen and elastin (weight ratio 1:1) with interconnected pores were prepared by freeze drying and crosslinked with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence or absence of a Jeffamine spacer (poly(propylene glycol)-bis-(2-aminopropyl ether), J230). The crosslinked and uncrosslinked matrices had porosities of 90% and average pore sizes of 131-151 microm. Smooth muscle cells (SMC) were cultured in the crosslinked and uncrosslinked tubular scaffolds under pulsatile flow conditions (mean flow rate 9.6 ml/min, 120 beats/min, pressure 80-120 mmHg). All the constructs could withstand cyclic mechanical strain in the absence of any mechanical support without cracking or suffering permanent deformation. After 7d, SMC were homogeneously distributed throughout the uncrosslinked and EDC/NHS crosslinked constructs, whereas hardly any cell was observed on the luminal side of J230/EDC/NHS crosslinked matrices. Considering the better mechanical performance of EDC/NHS crosslinked matrices compared to non-crosslinked constructs after 7d of culture, SMC were dynamically cultured in the former scaffolds for 14d. During this period, the high strain stiffness of the constructs increased more than two-fold to 38+/-2 kPa, whereas the low strain stiffness doubled to 8+/-2 kPa. The yield stress and yield strain were 30+/-10 kPa and 120+/-20%, respectively. SMC were homogeneously distributed throughout the EDC/NHS crosslinked collagen/elastin constructs and collagen fibres tended to orient in the circumferential direction.     

Enhanced biological stability of collagen with incorporation of PAMAM dendrimer

The crosslinking methods of collagen using glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) are frequently performed in biomedical applications, but both methods still have their own disadvantages, including the GTA cytotoxicity and low degree of EDC/NHS crosslinking. In this study, we incorporated polyamidoamine (PAMAM) dendrimer with surface amine groups into the two aforementioned crosslinking methods to improve the biostability and structural integrity of collagen. Fifty micromolar of dendrimer concentration was found to have negligible in vitro cytotoxicity and was used for EDC and GTA crosslinking of collagen. The collagenase digestion assay showed that the collagen scaffolds crosslinked in the presence of PAMAM exhibited a higher denature temperature and higher resistance against collagenase digestion compared with their counterparts without dendrimer. Cell proliferation with human conjunctival fibroblasts showed that the incorporation of PAMAM in EDC crosslinking significantly increased the proliferation. All the crosslinked scaffolds also exhibited higher structural stability than the noncrosslinked scaffold. Crosslinking with EDC and PAMAM together yielded substantially higher proliferation and may be a suitable collagen scaffold for biomedical applications.     

Design and Analysis of Braid-Twist Collagen Scaffolds

Collagen type I fiber-based scaffolds for anterior cruciate ligament (ACL) replacement were evaluated for their mechanical properties and their ability to promote cellular proliferation. Prior to scaffold formation, two crosslinking methods were investigated on individual reconstituted collagen type I fibers, ultraviolet radiation, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Crosslinking with EDC for 4 hr yielded mechanical properties similar to the human ACL; therefore, scaffold crosslinking was done with EDC for 4 hr. A braid-twist scaffold design was used, and scaffolds were left uncrosslinked, crosslinked after the addition of gelatin, or crosslinked without gelatin. The ultimate tensile strength, Young’s modulus, and viscoelastic properties of the scaffolds were then evaluated. In order to assess cellular response on the scaffolds, primary rat ligament fibroblast cells were seeded upon the scaffolds. Cell activity was evaluated at days 7, 14, and 21 using a Cell Titer 96® AQueous One Solution Cell Proliferation Assay (MTS Assay). The mechanical testing results showed that among the three scaffold groups, the crosslinked scaffolds without gelatin displayed an ultimate tensile strength, Young’s modulus, and viscoelastic properties that were closest to the human ACL. Improvements are still desired to enhance the mechanical compliance and ductility of these scaffolds. Cell activity was observed on all cell-seeded scaffolds by day 7, but by day 21 only the crosslinked scaffolds without gelatin displayed increased cellular activity compared with the negative controls. Although improvement is still needed, the results suggest that these scaffolds have the potential to contribute toward an ACL replacement strategy.     

Design and analysis of braid-twist collagen scaffolds

Collagen type I fiber-based scaffolds for anterior cruciate ligament (ACL) replacement were evaluated for their mechanical properties and their ability to promote cellular proliferation. Prior to scaffold formation, two crosslinking methods were investigated on individual reconstituted collagen type I fibers, ultraviolet radiation, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Crosslinking with EDC for 4 hr yielded mechanical properties similar to the human ACL; therefore, scaffold crosslinking was done with EDC for 4 hr. A braid-twist scaffold design was used, and scaffolds were left uncrosslinked, crosslinked after the addition of gelatin, or crosslinked without gelatin. The ultimate tensile strength, Young's modulus, and viscoelastic properties of the scaffolds were then evaluated. In order to assess cellular response on the scaffolds, primary rat ligament fibroblast cells were seeded upon the scaffolds. Cell activity was evaluated at days 7, 14, and 21 using a Cell Titer 96(?) AQueous One Solution Cell Proliferation Assay (MTS Assay). The mechanical testing results showed that among the three scaffold groups, the crosslinked scaffolds without gelatin displayed an ultimate tensile strength, Young's modulus, and viscoelastic properties that were closest to the human ACL. Improvements are still desired to enhance the mechanical compliance and ductility of these scaffolds. Cell activity was observed on all cell-seeded scaffolds by day 7, but by day 21 only the crosslinked scaffolds without gelatin displayed increased cellular activity compared with the negative controls. Although improvement is still needed, the results suggest that these scaffolds have the potential to contribute toward an ACL replacement strategy.     

EDC cross-linking improves skin substitute strength and stability

Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50mm). Cross-linking with EDC at concentrations >1mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10mm. At 50mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.     

ACL reconstruction using a novel hybrid scaffold composed of polyarylate fibers and collagen fibers

The objective was to perform an initial in vivo evaluation of a novel braided hybrid polyarylate and collagen fiber scaffold for the reconstruction of the anterior cruciate ligament (ACL). The braided hybrid scaffold is composed of 75% poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate)(12,10), [p(DTD DD)] fibers and 25% type I bovine collagen fibers. The scaffold is designed to temporarily bear mechanical loads and gradually degrade as neoligament tissue is deposited. Scaffolds were electron beam sterilized and used to reconstruct the ACL in five Finnish Dorset crossed-bred sheep in this feasibility study. At 4 (n = 1) and 12 (n = 4) weeks post-op, scaffolds were retrieved and analyzed for cellular ingrowth and strength retention. There was extensive cell infiltration and vascularity, which increased with time. Tissue ingrowth occurred throughout the cross section in the midsubstance of the scaffolds. After 12 weeks all scaffolds were intact. Femur-scaffold-tibia complex (FSTC) explanted at 12 weeks had a yield load of 42 ± 22 N and a stiffness of 9 ± 3 N mm(-1) . All scaffolds were well tolerated in the intraarticular space and induced tissue ingrowth, including new blood vessels, fibroblasts, inflammatory cells, and newly deposited collagen, throughout the cross section of the scaffold. Tissue ingrowth is critical to the success of a degradable scaffold for ACL reconstruction. Long-term studies in a large animal model are required to determine the efficacy of these novel hybrid scaffolds for ACL reconstruction. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2913-2920, 2012.     

First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.     

Mechanical loading of bovine pericardium accelerates enzymatic degradation.

Bioprosthetic heart valves fail as the result of two simultaneous processes: structural deterioration and calcification. Leaflet deterioration and perforation have been correlated with regions of highest stress in the tissue. The failures have long been assumed to be due to simple mechanical fatigue of the collagen fibre architecture; however, we have hypothesized that local stresses-and particularly dynamic stresses-accelerate local proteolysis, leading to tissue failure. This study addresses that hypothesis. Using a novel, custom-built microtensile culture system, strips of bovine pericardium were subjected to static and dynamic loads while being exposed to solutions of microbial collagenase or trypsin (a non-specific proteolytic enzyme). The time to extend to 30% strain (defined here as time to failure) was recorded. After failure, the percentage of collagen solubilized was calculated based on the amount of hydroxyproline present in solution. All data were analyzed by analysis of variance (ANOVA). In collagenase, exposure to static load significantly decreased the time to failure (P < 0.002) due to increased mean rate of collagen solubilization. Importantly, specimens exposed to collagenase and dynamic load failed faster than those exposed to collagenase under the same average static load (P = 0.02). In trypsin, by contrast, static load never led to failure and produced only minimal degradation. Under dynamic load, however, specimens exposed to collagenase, trypsin, and even Tris/CaCl2 buffer solution, all failed. Only samples exposed to Hanks' physiological solution did not fail. Failure of the specimens exposed to trypsin and Tris/CaCl2 suggests that the non-collagenous components and the calcium-dependent proteolytic enzymes present in pericardial tissue may play roles in the pathogenesis of bioprosthetic heart valve degeneration.     

Modulation of collagen proteolysis by chemical modification of amino acid side-chains in acellularized arteries

In this study, we have examined the effects of specific chemical modifications of amino acid side-chains on the in vitro degradation of "native" collagen obtained from acellular matrix (ACM)-processed arteries. Two monofunctional epoxides of different size and chemistry were used to modify lysine, with or without methylglyoxal modification of arginine. Biochemical, thermomechanical, tensile mechanical, and multi-enzymatic (collagenase, cathepsin B, acetyltrypsin, and trypsin) degradation analyses were used to determine the effects of modifications.Collagen solubilization by enzymes was found to depend upon the size and chemistry of epoxides used to modify lysine residues. In general, the solubilization of native ACM collagen by collagenase, cathepsin B, trypsin, and acetyltrypsin was either unaltered or decreased after modification with glycidol. In contrast, n-butylglycidylether (n-B) treatment increased solubilization by all enzymes. Subsequent arginine modification significantly reduced collagen solubilization by acetyltrypsin for glycidol-treated ACM arteries, whereas increased collagen solubilization was observed for n-B-treated ACM arteries with all enzymes. Gel chromatographic analyses of collagen fragments solubilized by trypsin revealed that both the amount and sites of cleavage were altered after lysine and arginine modification. The ability to modulate the enzymatic degradation of tissue-derived materials as demonstrated in this study may facilitate the design of novel engineering scaffolds for tissue regeneration or collagen-based drug delivery systems.     

Dendrimer crosslinked collagen as a corneal tissue engineering scaffold: mechanical properties and corneal epithelial cell interactions

Generation 2 polypropyleneimine octaamine dendrimers were used to generate highly crosslinked collagen with mechanical properties that would make it appropriate for use as a corneal tissue-engineering scaffold. Crosslinking of a highly concentrated collagen solution (2-4%) was effected using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC). The multifunctional dendrimers were introduced as novel multifunctional crosslinkers after the activation of the carboxylic acid groups of glutamic and aspartic acid residues in collagen. Glutaraldehyde, a common collagen crosslinker, was used as comparison, as was EDC, itself an alternative crosslinker, which forms "zero-length or self-crosslinking". The mechanical properties resultant gels were determined. Young's modulus of the dendrimer crosslinked gels was significantly higher than that observed with the other crosslinkers, increasing to 5 MPa compared with 0.1 MPa for the EDC crosslinked gels. Transmission electron microscopy (TEM) analysis of the gels demonstrated the presence of fibrils in the thermally gelled collagen controls; no fibrils were observed in the dendrimer crosslinked gels. As a result, the optical transparency of the dendrimer crosslinked collagen was significantly better than that of the collagen thermal gels. The EDC and glutaraldehyde crosslinked gels were generally less transparent than those crosslinked with the dendrimers. Glucose permeation results demonstrated that the dendrimer crosslinked collagen had higher glucose permeability than natural human cornea. Dendrimer crosslinked collagen gels supported human corneal epithelial cell growth and adhesion, with no cell toxicity. In comparison, some potentially cytotoxic effects were observed with glutaraldehyde crosslinked collagen. Overall, the dendrimer crosslinked collagen gels showed promising properties that suggest that these might be suitable scaffolds for corneal tissue engineering and potentially other tissue engineering applications.     

Fibroin/collagen hybrid hydrogels with crosslinking method: preparation, properties, and cytocompatibility

Substrate stiffness is an important physical factor in the response of many cell types. Although protein-based hydrogels are widely used as cell-culture substrates because of their resemblance to the natural extracellular matrix (ECM) and complex signaling, their rigidity should be further increased to facilitate the adhesion and growth of cells. In this study, fibroin/collagen hydrogels having suitable stiffness were prepared directly by adding 1-ethyl-3-(3-dimethylaminoprophy) carbodiimide hydrochloride (EDC) in fibroin/collagen solution to induce crosslinking. The storage moduli of these crosslinked hydrogels are above 3 kPa, and even exceed 10 kPa, having stronger mechanical strength than that of previously reported protein-based hydrogels. Furthermore, the crosslinked hydrogels can maintain their configuration above 80(o)C, which proves their increased thermal stability. Although crosslinked, the hydrogels still maintain the mobility of fibroin molecules. The growth of vascular smooth muscle cells (VSMCs) in fibroin/collagen gels indicates that the crosslinking reaction has no negative influence on the biocompatibility of fibroin/collagen hydrogels. The fibroin/collagen hydrogels are more propitious to the growth of cells compared with fibroin/collagen scaffolds. Because of their inherent biocompatibility, excellent mechanical and thermal properties, and green preparation process, the fibroin/collagen hydrogels would become promising scaffolds for tissue engineering.     

In vitro biomechanical testing of anterior cruciate ligament reconstruction: Traditional versus physiologically relevant load analysis

Various anterior cruciate ligament (ACL) graft-fixation devices exist. In this in vitro study a comparison of biomechanical characteristics of the Cross-Pin and button type fixation devices under practical rehabilitation loads was done. Forty bovine knees and hoof extensor tendons were harvested. After disarticulation, the femoral end of an ACL was prepared with either fixation, using the extensor tendon as graft. The mechanical test was either a single load to failure or load to failure after cycling loads. Twenty specimens were loaded to failure at a rate of 1 mm/s, remaining specimens were cycled between 50 and 250 N for 1000 cycles then failure tested in a similar manner. Results show that both forms of fixation are able to withstand loads that exceed those observed in performing functional activities. Activity-specific stiffness (loads comparable to walking, jogging and stair descent) was lower than linear stiffness for both EndoButton and Cross-Pin, without prior cycling. After cycling, activity-specific stiffness increased to linear stiffness values for the Cross-Pin for all activities. Thus, suggesting that the Cross-Pin provides a more rigid fixation after initial implantation over a wider range of activities, which would theoretically permit a more aggressive rehabilitation protocol and possibly an earlier return to regular activity. In contrast, activity-specific stiffness increased above linear stiffness values for the EndoButton only under heavier loads (jogging and stair descent). Dynamic stiffness was higher and displacement lower for Cross-Pin throughout the cycle test. These results indicate, in ACL reconstruction, that graft complex stiffness should be considered at relevant loads only.     

The effect of chemical modification of amino acid side-chains on collagen degradation by enzymes

In this study, the effects of specific chemical modifications of amino acid side-chains on the in vitro enzyme degradation of type I collagen was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine and methylglyoxal was used to modify arginine. Lysine residues were modified using glycidol, a small hydrophilic reagent or n-butylglycidylether, a larger hydrophobic reagent. Amino acid analysis, swelling measurements, in vitro enzyme degradation analyses (using either collagenase, trypsin, acetyltrypsin, or cathepsin B), and gel chromatography were used to determine the effects of each chemical modification on purified type I collagen. Collagen solubilization by enzymes depended upon the size and chemistry of epoxides used to modify lysine residues. Modification of lysine residues by glycidol and arginine modification by methylglyoxal together significantly reduced collagen solubilization by acetyltrypsin and collagenase, whereas increased collagen solubilization was observed for all enzymes after lysine modification with n-butylglycidylether combined with arginine modification by methylglyoxal. Gel chromatographic analyses of collagen fragments solubilized by acetyltrypsin from type I collagen revealed that both the extent of solubilization and sites of cleavage were altered after lysine and arginine modification. In contrast, lysine and arginine modification only altered the amount of collagen solubilized by collagenase and had no effect on the amount collagen solubilized by cathepsin B. The ability to modulate the enzyme degradation of collagen-based materials as demonstrated in this study may facilitate the design of novel scaffolds for tissue regeneration or collagen-based drug/protein/gene delivery systems.     

Synergistic effects of glucose and ultraviolet irradiation on the physical properties of collagen

Our objective was to strengthen and stabilize collagen films without the introduction of cytotoxic chemical crosslinkers. We hypothesized that collagen could be rapidly crosslinked with glucose with ultraviolet (UV) irradiation as a catalyst. In theory, UV-generated free radicals can expedite collagen crosslinking with glucose via the formation of reactive, linear glucose molecules. The mechanical properties of glucose-incorporated, UV-exposed collagen films and appropriate controls were determined under various conditions: (1) hydration in phosphate-buffered saline, (2) heat-denaturation, (3) incubation in a collagenase solution, and (4) incubation in a trypsin solution. Without exposure to UV, the incorporation of glucose into the films had no effect. Without glucose, exposure to UV increased the strength but caused significant denaturation. The combination of glucose and UV, however, synergistically improved the mechanical properties and enzyme resistance of collagen films, indicative of increased crosslinking without significant denaturation effects. The addition of thiourea, a potent free-radical scavenger, or aminoguanidine, an inhibitor of glucose-derived crosslinking, to the collagen films markedly hindered these synergistic effects. These data strongly suggest that free-radical-dependent, glucose-derived crosslinks provide the enhanced strength and enzyme resistance observed in glucose-incorporated, UV-exposed collagen films. Further studies are required to explore biomaterial applications of this novel collagen crosslinking method.     

Experimental validation of arthroscopic cartilage stiffness measurement using enzymatically degraded cartilage samples

In order to evaluate the ability of the arthroscopic indentation instrument, originally developed for the measurement of cartilage stiffness during arthroscopy, to detect cartilage degeneration, we compared changes in the stiffness with the structural and constitutional alterations induced by enzymes on the tissue in vitro. The culturing of osteochondral plugs on Petri dishes was initiated in Minimum Essential Medium with Earle's salts and the baseline stiffness was measured. Then, the experimental specimens were digested using 50 microg ml(-1) trypsin for 24 h, 0.1 U ml(-1) chondroitinase ABC or 30 U ml(-1) purified collagenase (type VII) for 24 h or 48 h (n = 8-15 per group). The control specimens were incubated in the medium. After the enzyme digestion, the end-point stiffness was measured and the specimens for the microscopic analyses were processed. The proteoglycan (PG) distribution was analysed using quantitative microspectrophotometry and the quantitative evaluation of the collagen network was made using a computer-based polarized light microscopy analysis. Decrease (p < 0.05) of cartilage stiffness was found after 24 h trypsin (36%) and 48 h chondroitinase ABC (24%) digestion corresponding to a decrease (p < 0.01) of up to 80% and up to 30% in the PG content respectively. Decrease of the superficial zone collagen content or arrangement (78%, p < 0.001) after 48 h collagenase digestion also induced a decrease (30%, p < 0.001) in cartilage stiffness. We conclude that our instrument is capable of detecting early structural and compositional changes related to cartilage degeneration.     

The susceptibility of hepatic collagen to homologous collagenase in human and experimental cirrhosis of the liver.

The susceptibility of hepatic collagen to homologous collagenase in human and experimental CCl4 cirrhosis of the liver has been explored in vitro by exposure of cryostat liver sections to the corresponding enzymes for different time periods. The morphology and extent of collagen degradation was studied by the Picrosirius red/polarizing microscopy technique. The results of various experiments indicate that collagen present in cryostat sections of both human and rat normal and cirrhotic livers is resistant to trypsin digestion for periods of exposure of up to 48 hours but that heating the sections to 60 C for 1 hour renders the collagen susceptible to degradation by trypsin. Incubation of cryostat liver sections with bacterial collagenase revealed progressive degradation of collagen with a uniform pattern of changes in the original color and diameter of the fibers. Exposure of liver sections to homologous collagenases gave rise to the same pattern of changes observed with bacterial collagenase, although less extensive when equal incubation periods were compared. Nevertheless, sufficiently prolonged incubation of liver sections with their homologous collagenases eventually showed degradation of all collagen present in all normal and cirrhotic liver sections. These observations suggest that in the presence of non rate-limiting concentrations of homologous collagenase, the susceptibility of hepatic collagen to the corresponding degrading enzyme is probably not responsible for the irreversibility of the disease.     

Neomycin enhances extracellular matrix stability of glutaraldehyde crosslinked bioprosthetic heart valves

Glutaraldehyde (GLUT) crosslinked porcine aortic heart valves are continued to be extensively used in heart valve replacement surgeries. GLUT does not crosslink glycosaminoglycans in the tissue and we have demonstrated that GAG loss is associated with tissue degeneration. In this study, we examined the ability of neomycin to enhance GLUT crosslinking to stabilize GAGs, as well as provide evidence of improved functional integrity. Neomycin enhanced GLUT crosslinked (NG) leaflets exposed to collagenase and elastase enzymes exhibited an increased resistance to proteolytic degradation. Furthermore, NG leaflets exhibited small but significant increases in collagen denaturation temperatures when compared to that of standard GLUT crosslinked BHVs. NG leaflets subjected to storage, accelerated cyclic fatigue, and in vitro enzyme mediated GAG degradation revealed improved GAG stabilization versus standard GLUT crosslinked valves, which sustained substantial decreases in GAG content. Ultrastructural analysis using transmission electron microscopy qualitatively confirmed NG leaflets preserved GAGs after enzymatic degradation. Biomechanical analyses demonstrated that NG leaflets were functionally similar to GLUT tissues but were slightly stiffer under both planar biaxial tension and under flexure. Interestingly, after GAGase treatment, GLUT tissues showed increased areal compliance and reduced hysteresis, while NG leaflets were unchanged. Collectively, NG cross-linking functionally insulated the tissue from GAG digestion, and imparted modest additional matrix stiffness but maintained tissue hysteresis properties.     

Crosslinked collagen hydrogels as corneal implants: Effects of sterically bulky vs. non-bulky carbodiimides as crosslinkers

We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.     

京尼平交联3D打印胶原/壳聚糖支架的表征北大核心

背景:胶原/壳聚糖支架需交联才能达到相应力学性能,有研究表示调节交联剂浓度可以在一定范围内调控支架的理化性能。目的:探究京尼平浓度对胶原/壳聚糖支架理化性能的影响,制备理化性能可调节的组织工程支架。方法:将胶原和壳聚糖粉末分别溶于弱酸后混合均匀,作为打印墨水,利用生物3D打印机低温打印胶原支架与胶原/壳聚糖支架,经冻干、中和处理后分别以1,3,5 mmol/L的京尼平进行交联。检测各组支架的表观结构稳定性、抗拉能力、溶胀性能、降解性能与生物相容性。结果与结论:①将支架在PBS中浸泡3 d后,对比未交联的冻干支架,交联后胶原支架表面维持规则的孔结构,但是支架出现明显变形;交联后胶原/壳聚糖支架表面结构规则,仅1 mmol/L京尼平交联的胶原/壳聚糖支架存在轻微变形。②随着京尼平浓度的增加,各组支架的力学性能增加,并且对应交联浓度下的胶原/壳聚糖支架力学性能好于胶原支架。③随着京尼平浓度的增加,胶原支架的溶胀率下降,胶原/壳聚糖支架的溶胀率无明显变化。④浸泡于胶原酶溶液中后,不同浓度京尼平交联的胶原支架在1 h内被完全降解,胶原/壳聚糖支架的降解速率随京尼平浓度的增加而降低,均呈现先快速后平缓的趋势。⑤将骨髓间充质干细胞接种于各组交联支架3 d后,1,3 mmol/L京尼平交联的胶原/壳聚糖支架(或胶原支架)上的细胞数量明显多于5 mmol/L京尼平交联的胶原/壳聚糖支架(P<0.05)。⑥结果表明,京尼平可在一定范围调节胶原/壳聚糖支架理化性能,其中3 mmol/L京尼平交联的胶原/壳聚糖支架具有较好的力学性能、抗酶解能力与生物相容性。