The impact of vacuum freeze-drying on collagen sponges after gas plasma sterilization

The sterilization of porous collagen sponges remains a challenging procedure. Gamma irradiation denatures collagen, resulting in dramatic changes to its structure. Ethylene oxide leaves toxic residues requiring weeks to evaporate. This study investigated the impact on cell behavior of gas plasma treatment when combined with vacuum freeze-drying. The goal of this procedure is to eliminate the molecules of hydrogen peroxide remaining after the sterilization process, together with their decomposition products, from the scaffolds. These molecules hinder the immediate use of the porous designs. Collagen and EDC/NHS-heparinized collagen scaffolds were sterilized with gas plasma. H2O2 released by the collagen specimens was measured by peroxidase test both immediately and also 1 week after the plasma treatment. Further measurements were done 24, 36, 48 and 72 h after vacuum freeze-drying. The activity of these scaffolds was further evaluated in relation to the proliferation, migration and differentiation of human umbilical vein endothelial cells (HUVECs). Both immediately after exposure to gas plasma and also 1 week later, the collagen designs contained significantly higher concentrations of H2O2 than scaffolds having also undergone vacuum freeze-drying. This procedure achieved faster decontamination of the remaining H2O2. Following vacuum freeze-drying, sponges already allowed HUVEC proliferation after 48 h, but in non-lyophilized specimens after gas plasma treatment alone, cell death occurred as early as only 1 week later. These data highlight the advantages of carrying out vacuum freeze-drying following gas plasma sterilization. The results show the substantial impact of sterilization of porous materials made for tissue engineering.     

Impact of sterilization on the porous design and cell behavior in collagen sponges prepared for tissue engineering

This study investigates the impact of different sterilization processes on structural integrity and stability of collagen sponges designed for tissue engineering. Collagen sponges with uniform pore size (20 microm) were sterilized either with ethylene oxide (EO) or gamma irradiation (2.5 Mrad). Gamma-sterilized sponges showed a dramatic decrease of resistance against enzyme degradation and severe shrinkage after cell seeding. Collapsed porosity inhibited fibroblasts and barred completely the human umbilical vein endothelial cell ingrowth into the sponges. On the contrary, the porous structure and stability of EO-sterilized sponges remained almost unaltered. Fibroblasts and endothelial cells exhibited favorable proliferation and migration within sponges with normal morphology. Tubular formation by seeded endothelial cells occurred early in the first week. Therefore, we emphasize that the impact of sterilization of biomaterials is substantial and any new procedure has to be evaluated by correlating the impact of the procedure on the porous structure with cell proliferation behavior.     

Effects of Low-Temperature Hydrogen Peroxide Gas Plasma Sterilization on In Vitro Cytotoxicity of Poly(ϵ-Caprolactone) (PCL)

Abstract

Our objective was to determine whether low-temperature hydrogen peroxide (H₂O₂) gas plasma sterilization of porous three-dimensional poly(?-caprolactone) (PCL) constructs significantly inhibits cellular metabolism of canine chondrocytes. Porous cylindrical constructs were fabricated using fused deposition modeling and divided into four sterilization groups. Two groups were sterilized with low-temperature H₂O₂ gas plasma (LTGP) and constructs from one of those groups were subsequently rinsed with Dulbecco’s Modified Essential Media (LTGPDM). Constructs in the other two groups were disinfected with either 70% isopropyl alcohol or exposure to UV light. Canine chondrocytes were seeded in 6-well tissue-culture plates and allowed to adhere prior to addition of PCL. Cellular metabolism was assessed by adding resazurin to the tissue-culture wells and assessing conversion of this substrate by viable cells to the fluorescent die resorufin. This process was performed at three times prior to addition of PCL and at four times after addition of PCL to the tissue-culture wells. Metabolism was not significantly different among the different tissue-culture wells at any of the 3 times prior to addition of PCL. Metabolism was significantly different among the treatment groups at 3 of 4 times after addition of PCL to the tissue culture wells. Metabolism was significantly lower with constructs sterilized by LTGP than all other treatment groups at all 3 of these times. We conclude that LTGP sterilization of PCL constructs resulted in significant cytotoxicity to canine chondrocytes when compared to PCL constructs disinfected with either UV light exposure or 70% isopropyl alcohol.     

Spatially Guided Angiogenesis by Three-Dimensional Collagen Scaffolds Micropatterned with Vascular Endothelial Growth Factor

Abstract

Successful regeneration of large and highly functionalized tissue and organs depends on the ability to guide blood vessel formation with three-dimensional scaffolds. Angiogenic growth factors have the potential to stimulate blood vessels in scaffolds. However, simply incorporating angiogenic growth factors in a random fashion may lead to uncontrolled blood vessel generation, which ultimately results in poor blood vessel network function and uneven growth of engineered tissue. To control and guide the formation of a blood vessel network in porous scaffolds, we prepared collagen sponges with micropatterned vascular endothelial growth factor (VEGF). VEGF was micropatterned in three-dimensional collagen sponges using micropatterned collagen/VEGF ice lines, which were prepared by a dispersing machine. The VEGF-micropatterned collagen sponges were implanted subcutaneously in nude mice. Following 6 weeks of implantation, the VEGF-micropatterned collagen sponges induced the formation of micropatterned blood vessel networks. More blood vessels were observed in the regions in which VEGF was immobilized than those without VEGF. The micropattern of VEGF determined the micropattern of the regenerated blood vessel network. The spatial immobilization of VEGF in three-dimensional porous scaffolds may be useful to stimulate guided blood vessel formation in a variety of tissue-engineering applications.     

The role of histamine in wound healing. II. The effect of antagonists and agonists of histamine receptors (H1 and H2) on collagen levels in granulation tissue

The effects of mepyramine (H₁-receptor antagonist), cimetidine (H₂-receptor antagonist), IEM-813 (H₁-receptor agonist) and 4-Met-Hi (H₂-receptor agonist) on collagenogenesis processes in subcutaneous implanted sponges of rats were studied. Administration into sponges of cimetidine 30 min before histamine injection blocked both the stimulatory effect of low histamine doses and the inhibitory effect of high histamine doses on collagen formation. Low doses of 4-Met-Hi increased collagen levels but reduced the levels at high doses. Mepyramine and IEM-813 did not influence collagen biosynthesis.     

Collagen microgel-assisted dexamethasone release from PLLA-collagen hybrid scaffolds of controlled pore structure for osteogenic differentiation of mesenchymal stem cells

Directed stem cell differentiation over three-dimensional porous scaffolds capable of releasing bioactive instructive cues is an important tool in tissue engineering. In this research, we have prepared dexamethasone (Dex)-releasing collagen microbead-functionalized poly(L-Lactide)-collagen hybrid scaffolds as an osteoinductive platform for human bone marrow-derived mesenchymal stem cells (MSCs). The scaffolds were prepared by a combined method of emulsion freeze-drying and porogen-leaching using pre-prepared ice collagen particulates as a porogen material. Dex release from the hybrid scaffolds was studied at 37?°C under shaking condition and the impact of released Dex towards osteogenic lineage differentiation was investigated by 3?week in vitro culture of MSCs. The results showed that hybrid scaffolds had controlled pore structure and interconnected pores deposited with collagen fibers. The hybrid scaffold facilitated cell seeding and the spatial localization of Dex/collagen microbeads facilitated a microgel-assisted spatio-temporal control of Dex release. The released Dex was useful for osteogenic differentiation of MSCs, which was confirmed from the elevated expression of osteogenic-specific gene-encoded proteins. The hybrid scaffolds should be useful for regeneration of a functional bone tissue.     

Porosity and biological properties of polyethylene glycol-conjugated collagen materials

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.     

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.     

Development of Porous Alginate-Based Scaffolds Covalently Cross-Linked through a Peroxidase-Catalyzed Reaction

Porous scaffolds are important in tissue engineering. We developed porous scaffolds from the hydrogels of an alginate derivative bearing phenolic hydroxyl groups. The hydrogels were prepared using horseradish peroxidase (HRP) to catalyze the cross-linking between the phenolic hydroxyl groups. A porous structure with a pore size of approx. 200 μm was developed through simultaneous water-extraction and ionic cross-linking by calcium ions by soaking frozen hydrogels in the mixture of ethanol and CaCl₂ solution at –20°C. Due to the existence of the covalent cross-links developed through the enzymatic reaction, the porous form had a higher stability from a loss of cross-linked calcium ions than that obtained from non-modified sodium alginate (Na-Alg). The porous specimen developed from the hydrogel obtained with 10 U/ml HRP and 10 mM H₂O₂ showed about 1.5-times greater repulsion forces than those detected for the porous specimen obtained from Na-Alg toward compressions. No harmful effects of the enzymatically cross-linked specimens were detected on the growth and morphology of the entrapped cells: cells in the enzymatically cross-linked specimens showed almost the same growth profile and morphology with those in the porous specimen obtained from Na-Alg.     

Synthesis and characterization of hierarchically macroporous and mesoporous CaO-MO-SiO(2)-P(2)O(5) (M=Mg, Zn, Sr) bioactive glass scaffolds

Mg-, Zn- and Sr-doped hierarchically macroporous and mesoporous CaO–MO–SiO₂–P₂O₅ (M = Mg, Zn or Sr) bioactive glass (HMMBG) scaffolds were synthesized using the non-ionic block copolymer EO₂₀PO₇₀EO₂₀ and polyurethane sponges as cotemplates. The Mg-, Zn- or Sr-doped HMMBG scaffolds showed no distinct difference in phase composition, macroporous structure or pore volume from the HMMBG scaffolds without Mg, Zn or Sr. The Mg-, Zn- and Sr-doped HMMBG scaffolds showed no cytotoxicity. The gradual release of Ca, P, Si, Mg, Zn and Sr into the culture medium from these scaffolds contributed to the enhancement of the proliferation and ALP activity of mesenchymal stem cells (MSCs). The Mg-, Zn- and Sr-doped HMMBG scaffolds may be used as bone substitute materials.     

Nature-derived epigallocatechin gallate/duck’s feet collagen/hydroxyapatite composite sponges for enhanced bone tissue regeneration

Abstract

Scaffolds mimicking structural and chemical characteristics of the native bone tissues are critical for bone tissue engineering. Herein, we have developed and characterized epigallocatechin gallate/duck’s feet collagen/hydroxyapatite (EGCG/DC/HAp) composite sponges that enhanced the bone tissue regeneration. The three-dimensional composite sponges were synthesized by loading various amounts (i.e. 1, 5 and 10 μM) of EGCG to duck feet derived collagen followed by freeze-drying and then coating with hydroxyapatite. Several measuremental techniques were employed to examine the properties of the as-fabricated composite sponges including morphology and structure, porosity, compressive strength, etc. and as well compared with pristine duck feet derived collagen. SEM observations of EGCG/DC/HAp sponges showed the formation of a highly porous collagen matrix with EGCG embodiment. The porosity and pore size of sponges were found to increase by high EGCG content. The compressive strength was calculated as 3.54 ± 0.04, 3.63 ± 0.03, 3.89 ± 0.05, 4.047 ± 0.05 MPa for 1, 5 and 10 μM EGCG/DC/HAp sponges, respectively. Osteoblast-like cell (BMSCs isolated from rabbit) culture and in vivo experiments with EGCG/DC/HAp sponges implanted in nude mouse followed by histological staining showed enhanced cell internalization and attachment, cell proliferation, alkaline phosphatase expressions, indicating that EGCG/DC/HAp sponges have ahigh biocompatibility. Moreover, highEGCG content in the EGCG/DC/HAp sponges have led to increased cellular behavior. Collectively, the 5 μM of EGCG/DC/HAp sponges were suggested as the potential candidates for bone tissue regeneration.     

Water/O2-Plasma-Assisted Treatment of PCL Membranes for Biosignal Immobilization

The main purpose of this study was to obtain COOH functionalities on the surface of poly-ε-caprolactone (PCL) membranes using low-pressure water/O₂-plasma-assisted treatment. PCL membranes were prepared using the solvent-casting technique. Then, low-pressure water/O₂ plasma treatments were performed in a cylindrical, capacitively coupled RF-plasma-reactor in three steps: H₂O/O₂-plasma treatment; in situ (oxalyl chloride vapors) gas/solid reaction to convert –OH functionalities into –COCl groups; and hydrolysis for final –COOH functionalities. Optimization of plasma modification processes was done using the DoE software program. COOH and OH functionalities on modified surfaces were detected quantitatively using the fluorescent labeling technique and an UVX 300G sensor. Chemical structural information of untreated, plasma treated and oxalyl chloride functionalized PCL membranes were acquired using pyrolysis GC/MS and ESCA analysis. High-resolution AFM images revealed that nanopatterns were more affected than micropatterns by plasma treatments. AFM images recorded with amino-functionalized tips presented increased size of the features on the surface that suggests higher density of the carboxyls on the nanotopographical elements. Low-pressure water/O₂-plasma-treated and oxalyl chloride functionalized samples were biologically activated with insulin and/or heparin biosignal molecules using a PEO (polyoxyethylene bis amine) spacer. The success of the immobilization process was checked qualitatively by ESCA analysis. In addition, fluorescent labeling techniques were used for the quantitative determination of immobilized biomolecules. Cell-culture experiments indicated that biomolecule immobilization onto PCL scaffolds was effective on L929 cell adhesion and proliferation, especially in the presence of heparin.     

Acceleration of bone formation with BMP2 in frame-reinforced carbonate apatite–collagen sponge scaffolds

The development is expected of scaffold biomaterials that feature a shape-maintaining property in addition to high porosity and large pores that cells can easily invade. To develop a new biodegradable scaffold biomaterial reinforced with a frame, synthesized carbonate apatite (CO₃Ap) was mixed with neutralized collagen gel, and the CO₃Ap–collagen mixtures were lyophilized into sponges in a porous hydroxyapatite (HAp) frame ring. X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) analyses together with chemical analysis indicated that the synthesized CO₃Ap had a crystalline nature and a chemical composition similar to that of bone. Scanning electron microscope (SEM) observation showed that the CO₃Ap–collagen sponge had a sui pore size for cell invasion. In proliferation and differentiation experiments with osteoblasts, alkaline phosphatase and osteopontin activity were clearly detected. When these sponge–frame complexes with bone morphogenic protein (rh-BMP2) were implanted beneath the periosteum cranii of rats, significant new bone was created at the surface of the periosteum cranii after 4 weeks of implantation. These reinforced CO₃Ap–collagen sponges with rh-BMP2 are expected to be used as hard tissue scaffold biomaterials for the therapeutic purpose of the rapid cure of bone defects.     

Quasi-total-body exposure to an oxygen-ozone mixture in a sauna cabin

We have investigated the effects of quasi-total-body exposure of healthy volunteers to either an oxygen-ozone mixture (O₂-O₃) or to oxygen (O₂) alone during a short period in a sauna cabin. The subjects underwent both an experimental and a control examination, separated by a 3.5-month interval. Body mass, blood pressure, body temperature changes, electrocardiograms, venous blood gas and haemocytometric analyses, total antioxidant status and plasma levels of protein thiol groups, thiobarbituric acid reactive substances (TBARS), plasma cytokine, hepatic enzymes and creatine were determined before, immediately after the 20-min period in the cabin and then 0.5, 1.0 and 24?h afterwards. We observed statistically significant variations of body temperature, venous partial pressure of O₂ values, TBARS and plasma levels of interleukin 8, particularly after O₂-O₃ exposure. The increase in TBARS plasma levels concomitant with protein oxidation has been tentatively interpreted as being attributable to the transcutaneous passage of some reactive O₂ species, which should be considered if this approach is to be used as a biological response modifier. However, in the present study no adverse effects were noted after one session.     

In vitro contraction rate of collagen in sponge-shape matrices

Connective tissue substitute can be made of collagen sponge-shape matrice which is reconstituted by freeze-drying a collagen dispersion. This procedure is then followed by a crosslinking treatment to decrease the in vivo biodegradation rate. In the present study, collagen dispersions made of collagen fibrils with a D-staggered pattern were submitted to the following treatments: (1) cyanamide or glutaraldehyde was introduced during the dispersion step followed by the manufacture of sponges; (2) uncrosslinked sponges were exposed to formaldehyde vapor; or (3) uncrosslinked and crosslinked sponges were severely dehydrated. To characterize the in vitro contraction rate, the surface areas of sponges were sequentially recorded in relation to soaking time. Contraction did not significantly occur when sponges were chemically treated. However, collagen in sponges treated by either severe dehydration or by both cyanamide treatment and severe dehydration contracted. On the other hand, the different treatments of the collagen modified the distribution of the D-staggered pattern within fibrils. After glutaraldehyde treatment, the periodicity of collagen fibrils disappeared and large fibres were observed. These experiments show that the different treatments of the collagen can be useful for designing a contractile as well as a non-contractile biomaterial.     

In vitro contraction rate of collagen in sponge-shape matrices.

Connective tissue substitute can be made of collagen sponge-shaped matrice which is reconstituted by freeze-drying a collagen dispersion. This procedure is then followed by a crosslinking treatment to decrease the in vivo biodegradation rate. In the present study, collagen dispersions made of collagen fibrils with a D-staggered pattern were submitted to the following treatments: (1) cyanamide or glutaraldehyde was introduced during the dispersion step followed by the manufacture of sponges; (2) uncrosslinked sponges were exposed to formaldehyde vapor; or (3) uncrosslinked and crosslinked sponges were severely dehydrated. To characterize the in vitro contraction rate, the surface areas of sponges were sequentially recorded in relation to soaking time. Contraction did not significantly occur when sponges were chemically treated. However, collagen in sponges treated by either severe dehydration or by both cyanamide treatment and severe dehydration contracted. On the other hand, the different treatments of the collagen modified the distribution of the D-staggered pattern within fibrils. After glutaraldehyde treatment, the periodicity of collagen fibrils disappeared and large fibres were observed. These experiments show that the different treatments of the collagen can be useful for designing a contractile as well as a non-contractile biomaterial.     

Tailoring the morphology of high molecular weight PLLA scaffolds through bioglass addition

Thermally induced phase separation (TIPS) has proven to be a suitable method for the preparation of porous structures for tissue engineering applications, and particular attention has been paid to increasing the pore size without the use of possible toxic surfactants. Within this context, an alternative method to control the porosity of polymeric scaffolds via the combination with a bioglass is proposed in this work. The addition of a bioactive glass from the 3CaO·P₂O₅–MgO–SiO₂ system enables the porous structure of high molecular weight poly(l-lactic) acid (PLLA) scaffolds prepared by TIPS to be tailored. Bioglass acts as a nucleating catalyst agent of the PLLA matrix, promoting its crystallization, and the glass solubility controls the pore size. A significant increase in the pore size is observed as the bioglass content increases and scaffolds with large pore size (～150 μm) can be prepared. In addition, the bioactive character of the scaffolds is proved by in vitro tests in synthetic plasma. The importance of this approach resides on the combination of the ability to tailor the porosity of polymeric scaffolds via the tunable solubility of bioglasses, without the use of toxic surfactants, leading to a composite structure with suitable properties for bone tissue engineering applications.     

Collagen–hyaluronic acid scaffolds for adipose tissue engineering

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen–hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity ～85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen–HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen–HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen–HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen–HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen–HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.     

Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H₂O₂) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O₂, normobaric hyperoxia (0.95 ATA O₂) or hyperbaric hyperoxia (HBO₂, 3.25 ATA O₂) for 60 min. H₂O₂ (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H₂O₂. Average membrane roughness (R_a) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R_a of the plasma membrane was 34±3, 57±3 and 63±5 nm in 0.20 ATA O₂, 0.95 ATA O₂ and HBO₂, respectively. R_a was 56±7 and 138±14 nm in 0.2 and 2 mM H₂O₂. Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H₂O₂ and correlated with O₂-induced membrane blebbing (r²=0.93). Coapplication of antioxidant, Trolox-C (150 μM), significantly reduced membrane R_a and MDA levels during hyperoxia. Hyperoxia-induced H₂O₂ production increased 189%±5% (0.95 ATA O₂) and 236%±5% (4 ATA O₂) above control (0.20 ATA O₂). We conclude that MLP and membrane blebbing increase with increasing O₂ concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.     

Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.