Type I Collagen Immobilized Poly(caprolactone) Nanofibers: Characterization of Surface Modification and Growth of Fibroblasts

Poly(caprolactone) (PCL) electrospun nanofibers were modified by aminolysis and collagen was immobilized on the aminolysed PCL nanofibers. Considering low immunogenic response collagen elicits, immobilization of the same is anticipated to enhance the tissue engineering application of the PCL nanofibers. Amino groups were introduced into PCL nanofibers through aminolysis process. Aminolysis of PCL nanofibers was confirmed by electron dispersive X‐ray analysis (EDX). Collagen was immobilized on aminolysed PCL nanofibers using glutaraldehyde as crosslinker. The collagen crosslinking on to PCL nanofibers was established by attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy. The fiber morphologies of PCL nanofibers at different stages were characterized by scanning electron microscopy (SEM). The change in hydrophobicity of PCL nanofibers due to aminolysis and collagen immobilization was determined by water contact angle measurements. Aminolysis followed by collagen immobilization had reduced the intrinsic hydrophobicity of PCL nanofibers. NIH 3T3 fibroblasts were cultured for 2 days on PCL nanofibers, aminolysed PCL nanofibers, and aminolysed PCL nanofibers crosslinked with collagen. Cell attachment and growth were observed by MTT assay in each case. Collagen immobilization improved the biocompatibility of the PCL nanofibers. Thus the modified PCL nanofibers can be used as suitable broad spectrum scaffold for skin, cartilage, bone, cardiac constructs for efficient tissue engineering applications.     

Mechanically induced structural changes during dynamic compression of engineered cartilaginous constructs can potentially explain increases in bulk mechanical properties

Several studies on chondrocyte-seeded hydrogels in bioreactor culture report increased mechanical properties of mechanically loaded constructs compared with unloaded free swelling controls despite no significant differences in biochemical composition. One possible explanation is that changes in the collagen architecture of dynamically compressed constructs lead to improved mechanical properties. Collagen molecules are incorporated locally into the extracellular matrix with individual stress-free configurations and orientations. In this study, we computationally investigated possible influences of loading on the collagen architecture in chondrocyte-seeded hydrogels and their resulting mechanical properties. Both the collagen orientation and its stress-free configuration were hypothesized to depend on the local mechanical environment. Reorientation of the collagen network alone in response to dynamic compression leads to a prediction of constructs with lower compressive properties. In contrast, remodelling of the stress-free configuration of the collagen fibres was predicted to result in a more compacted tissue with higher swelling pressures and an altered pre-stressed state within the collagen network. Combining both mechanisms resulted in predictions of construct geometry and mechanical properties in agreement with experimental observations. This study provides support for the hypothesis that structural changes to the collagen network contribute to the enhanced mechanical properties of cartilaginous tissues engineered in bioreactors.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.     

Engineering cartilage tissue interfaces using a natural glycosaminoglycan hydrogel matrix — An in vitro study

Hydrogels are suitable matrices for cartilage tissue engineering on account of their resemblance to native extracellular matrix of articular cartilage and also considering its ease of application, they can be delivered to the defect site in a minimally invasive manner. In this study, we evaluate the suitability of a fast gelling natural biopolymer hydrogel matrix for articular cartilage tissue engineering. A hydrogel based on two natural polymers, chitosan and hyaluronic acid derivative was prepared and physicochemically characterized. Chondrocytes were then encapsulated within the hydrogel and cultured over a period of one month. Cartilage regeneration was assessed by histological, biochemical and gene expression studies. Chondrocytes maintained typical round morphology throughout the course of this investigation, indicating preservation of their phenotype with sufficient production of extracellular matrix and expression of typical chondrogenic markers Collagen type 2 and aggrecan. The results suggest that the natural polymer hydrogel matrix can be used as an efficient matrix for articular cartilage tissue engineering.     

The regeneration of articular cartilage using a new polymer system

A polymer system based on room temperature polymerising poly (ethylmethacrylate) polymer powder and tetrahydrofurfuryl monomer has been investigated as a biomaterial for encouraging articular cartilage repair. This heterocyclic methacrylate polymer system swells slightly in situ and thus provides a good interface with subchondral bone resulting in mechanical stability with favourable uptake kinetics. Another feature of this polymer system is that it exhibits high water uptake which leads to absorption of the surrounding tissue fluid and matrix proteins, including growth factors; this may encourage the formation of new cartilage. Three weeks after implantation the tissue overgrowth contained cartilage components: chondrocytes, collagen type II, chondroitin 4-sulphate and chondroitin 6-sulphate. In addition numerous chondrocyte clones were observed at the edge of the defect and in the newly repaired tissue. By six weeks a superficial articulating surface was continuous with the normal articular cartilage with underlying tissue which showed some evidence of endochondral ossification. By nine weeks the surface covering of new cartilage had a widened and an irregular zone of calcified cartilage with thickened subchondral bone was present. At eight months the resurfaced cartilage remained intact above a remodelled subchondral bone end plate.     

EDC/NHS-crosslinked type II collagen-chondroitin sulfate scaffold: characterization and in vitro evaluation

Three-dimensional biodegradable porous type II collagen scaffolds are interesting materials for cartilage tissue engineering. This study reports the preparation of porous type II collagen-chondroitin sulfate (CS) scaffold using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The physico-chemical properties and ultrastructural morphology of the collagen scaffolds were determined. Then, isolated chondrocytes were cultured in porous type II collagen scaffolds either in the presence and/or absence of covalently attached CS up to 14 days. Cell proliferation, the total amount of proteoglycans and type II collagen retained in the scaffold and chondrocytes morphology were evaluated. The results suggest that EDC-crosslinking improves the mechanical stability of collagen-CS scaffolds with increasing EDC concentration. Cell proliferation and the total amount of proteoglycans and type II collagen retained in the scaffolds were higher in type II collagen-CS scaffolds. Histological analysis showed the formation of a denser cartilaginous layer at the scaffold periphery. Scanning electron microscopy (SEM) revealed chondrocytes distributed the porous surface of both scaffolds maintained their spherical morphology. The results of the present study also indicate that type II collagen-CS scaffolds have potential for use in tissue engineering.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

Collagen/hydroxyapatite composite obtained by electric field orientation

The purpose of this study was to obtain and characterize the oriented collagen/hydroxyapatite (COLL/HA) composite materials, using different kind of electric field. The orientation process takes place due to the dipolar collagen structure. The collagen molecules are electrically charged and can become oriented if an electric field is applied. The composites were obtained by the co-precipitation method starting from collagen gel and hydroxyapatite precursor. The mineralized collagen gel was introduced in an electric field for 1 h, frozen and lyophilized. In order to study the orientation process parameters, two types of electric fields were tested: pulsatory and superposed (both direct and pulsatory). The obtained materials were characterized by XRD, FTIR and SEM. Best results in what it concerns the oriented character of the microstructures, as resulted from SEM images, were obtained for a pulsatory field of 0.93 V/cm, the presence of the direct electric field component decreasing the orientation degree. The average orientation degree, calculated at the magnification of 1000× was about 95%.     

Structure–mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model

The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry.     

Influence of alginate on type II collagen fibrillogenesis

Collagen II is the majority of extracellular matrix components in articular cartilage, which with the major functions of preventing expansion of the tissue and distributing the load of body weight. To obtain man-made ECM, the reconstitution of collagen could be conducted in the presence of negatively charged polysaccharide, such as alginate. Alginate is an anionic polysaccharide capable of eversible gelated in calcium ion solution to prepare different shapes of biomaterials. Its well-known biocompatibility makes it an ideal material in biomedical applications. Thus, the aim of this study was to evaluate the effects of alginate on the fibrillogenesis of type II collagen. The preliminary results revealed that inclusion of alginate into soluble type II collagen solution could inhibit the development of turbidity of collagen solution, and the apparent rate constants in lag and growth phases decreased during collagen formation period, both rate constants decreased to about one-third of the original constants, respectively. From TEM observations, the collagen fibrils were significantly thicker in 0.05% and 0.1% alginate as compared with pure collagen solution. Furthermore, the D-periods of collagen fibers kept unchanged significantly under all reconstituted conditions, which meant the packing of collagen monomer was probably not affected by adding these amounts of alginate.     

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.     

The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1)

The aim of this study was to investigate the degree of deacetylation (DD) and molecular weight (MW) of chitosan within chitosan–collagen scaffolds on mouse osteoblasts (MC3T3-E1). The chitosan–collagen scaffolds were fabricated by freeze-drying technique. The studies on cell attachment and proliferation, alkaline phosphatase (ALP) activity, cell morphology, and mineralized nodule formation by osteoblasts on scaffolds were investigated. No statistically significant difference was found on cell attachment, but the chitosan–collagen scaffolds with low-DD chitosan had a statistically significantly (P < 0.05) higher proliferative effect and ALP activity than those scaffolds with high-DD chitosan, regardless of molecular weight. Scanning electron images demonstrated that MC3T3-E1 cells grew well on all test scaffolds; on the contrary, mineralized nodule formation was not found. In conclusion, the DD of chitosan is a crucial factor for MC3T3-E1 cells and it should be considered in further applications for bone tissue engineering.     

Formulation of PEG-based hydrogels affects tissue-engineered cartilage construct characteristics

The limited supply of cartilage tissue with appropriate sizes and shapes needed for reconstruction and repair has stimulated research in the area of hydrogels as scaffolds for cartilage tissue engineering. In this study we demonstrate that poly(ethylene glycol) (PEG)-based semi-interpenetrating (sIPN) network hydrogels, made with a crosslinkable poly(ethylene glycol)-dimethacrylate (PEGDM) component and a non-crosslinkable interpenetration poly(ethylene oxide) (PEO) component, and seeded with chondrocytes support cartilage construct growth having nominal thicknesses of 6 mm and relatively uniform safranin-O stained matrix when cultured statically, unlike constructs grown with prefabricated macroporous scaffolds. Even though changing the molecular weight of the PEO from 100 to 20 kDa reduces the viscosity of the precursor polymer solution, we have demonstrated that it does not appear to affect the histological or biochemical characteristics of cartilaginous constructs. Extracellular matrix (ECM) accumulation and the spatial uniformity of the ECM deposited by the embedded chondrocytes decreased, and hydrogel compressive properties increased, as the ratio of the PEGDM:PEO in the hydrogel formulation increased (from 30:70 to 100:0 PEGDM:PEO). Total collagen and glycosaminoglycan contents per dry weight were highest using the 30:70 PEGDM:PEO formulation (24.4+/-3.5% and 7.1+/-0.9%, respectively). The highest equilibrium compressive modulus was obtained using the 100:0 PEGDM:PEO formulation (0.32+/-0.07 MPa), which is similar to the compressive modulus of native articular cartilage. These results suggest that the versatility of PEG-based sIPN hydrogels makes them an attractive scaffold for tissue engineering of cartilage.     

Decyl bis phosphonate–protein surface modification of Ti–6Al–4V via a layer-by-layer technique

Titanium and its alloys have been applied in orthopedics due to their biocompatibility, mechanical, and physical properties. Here we use decyl bis phosphonate (DBP) and collagen I to modify Ti–6Al–4V through layer-by-layer technique in order to improve its bioactivity. The abilities of bovine serum albumin (BSA) adsorption and biomimetic mineralization of different sample surfaces were studied. X-ray photoelectron spectroscopy (XPS) and water contact angle data showed that DBP and collagen I were assembled on substrates successfully. The absorbance of BSA solution acquired from ultraviolet spectrophotometer (UV) indicated that samples of Ti–6Al–4V/DBP/Collagen and Ti–6Al–4V/DBP/Collagen/DBP adsorbed BSA most, followed by Ti–6Al–4V/DBP and Ti–6Al–4V. Scanning electron microscope (SEM) photos and X-ray diffraction (XRD) data showed that sample of Ti–6Al–4V/DBP/Collagen had better bioactivity in inducing HA formation than other samples tested in this investigation.     

Biochemical markers of the mechanical quality of engineered hyaline cartilage

The aim of this study was to determine whether or not biochemical markers can be used as surrogate measures for the mechanical quality of tissue engineered cartilage. The biochemical composition of tissue engineered cartilage constructs were altered by varying either (i) the initial cell seeding density of the scaffold (seeding density protocol) or (ii) the length of time the engineered tissue was cultured (culture period protocol). The aggregate or Young’s moduli of the constructs were measured (by confined or unconfined compression respectively), and compared with the composition of the extracellular matrix by quantitative measurement of the glycosaminoglycan (GAG), hydroxyproline, collagen I and collagen II and collagen cross-links. The aggregate modulus correlated positively with both GAG and collagen II content, but not with collagen I content. Young’s modulus correlated positively with GAG, collagen II and collagen I content, and the ratio of mature to immature cross-links. There was no significant correlation of Young’s Modulus with total collagen measured as hydroxyproline content. These results suggested that hydroxyproline determination may be an unreliable indicator of mechanical quality of tissue engineered cartilage, and that a measure of collagen II and GAG content is required to predict the biomechanical quality of tissue engineered cartilage.     

Fibril density reduction in keratoconic corneas

This study aims to estimate the reduction in collagen fibril density within the central 6 mm radius of keratoconic corneas through the processing of microstructure and videokeratography data. Collagen fibril distribution maps and topography maps were obtained for seven keratoconic and six healthy corneas, and topographic features were assessed to detect and calculate the area of the cone in each keratoconic eye. The reduction in collagen fibril density within the cone area was estimated with reference to the same region in the characteristic collagen fibril maps of healthy corneas. Together with minimum thickness and mean central corneal refractive power, the cone area was correlated with the reduction in the cone collagen fibrils. For the corneas considered, the mean area of keratoconic cones was 3.30 ± 1.90 mm². Compared with healthy corneas, fibril density in the cones of keratoconic corneas was lower by as much as 35%, and the mean reduction was 17 ± 10%. A linear approximation was developed to relate the magnitude of reduction to the refractive power, minimum corneal thickness and cone area (R² = 0.95, p < 0.001). Outside the cone area, there was no significant difference between fibril arrangement in healthy and keratoconic corneas. The presented method can predict the mean fibril density in the keratoconic eye''s cone area. The technique can be applied in microstructure-based finite-element models of the eye to regulate its stiffness level and the stiffness distribution within the areas affected by keratoconus.     

Mineralization of Collagen‐Coated Electrospun Poly(lactide‐co‐glycolide) Nanofibrous Mesh to Enhance Growth and Differentiation of Osteoblasts and Bone Marrow Mesenchymal Stem Cells

To obtain the biomimetic scaffolding materials for bone tissue engineering, poly(lactide‐co‐glycolide) (PLGA) nanofibrous mesh (NFM) was mineralized in a 5× simulated body fluid (SBF) for different time after it was treated by air plasma for 15 min and subsequent collagen coating. The apatite particles were nucleated on the surface of individual nanofibers, gradually grew up, and finally covered the whole NFM surface. The mineral aggregates were mainly composed of tiny hydroxyapatite (HA) nanoparticles, whose content reached a constant value of 54 µg · cm^?2 after 9 days. The collagen coating and apatite deposition enhanced the NFM strength pronouncedly too. In vitro cell culture demonstrated that the non‐ or less mineralized NFMs were more beneficial of cell spreading and proliferation than those highly mineralized NFMs, but the latter ones could strongly promote secretion of alkaline phosphatase (ALP) by osteoblasts after cultured for 14 days. Moreover, the highly mineralized NFMs also could significantly up‐regulated ALP activity and calcium synthesis of bone marrow mesenchymal stem cells (BMSCs), demonstrating that these NFMs are more favorable of the osteoblast phenotype expression and osteogenic induction. Therefore, the biomimetic apatite deposited PLGA/collagen NFM could be a promising candidate scaffold for bone tissue engineering.     

Correlating changes in collagen secondary structure with aging and defective type II collagen by Raman spectroscopy

A novel application of Raman spectroscopy for monitoring damage to ocular collagen in wild-type mice and Del1 (+/-) transgenic mice, a murine animal model of osteoarthritis, is described. In order to understand the progression of diseases of collagen, it is necessary to use methods that can recognize alterations in affected tissue due to chemical and/or genetic modifications. The heterozygous Del1 (+/-) transgenic mouse is established as a model for early-onset osteoarthritis caused by modifications to the type II collagen gene (COL2A1) that result in a truncated collagen fiber. We expect that abnormal type II collagen is expressed in articular cartilage and eye tissue of the Del1 (+/-) mouse. Eyes excised from a subset of specimens from another study using Del1 (+/-) mice were examined by Raman spectroscopy for evidence of defective collagen. Spectral contributions from the collagen protein were readily observed. The amide III envelope (1220-1280 cm-1) was used to characterize changes in collagen secondary structure. Raman spectra of the sclera component of eyes taken from transgenic and older wild-type mice show an increased collagen disorder, as expected. These preliminary results suggest that Raman is capable of recognizing and measuring abnormality in eye collagen and may have potential as a diagnostic tool for ocular collagen damage.     

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.