Michael D. Buschmann

Novel injectable neutral solutions of chitosan form biodegradable gels in situ

A novel approach to provide, thermally sensitive neutral solutions based on chitosan/polyol salt combinations is described. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells and therapeutic proteins; they form monolithic gels at body temperature. When injected in vivo the liquid formulations turn into gel implants in situ. This system was used successfully to deliver biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications. This study reports for the first time the use of polymer/polyol salt aqueous solutions as gelling systems, suggesting the discovery of a prototype for a new family of thermosetting gels highly compatible with biological compounds.

A validated 1H NMR method for the determination of the degree of deacetylation of chitosan

A method for the determination of the degree of deacetylation (DDA) of chitosan by 1H NMR spectroscopy has been formally validated. Chitosans with DDA ranging from 48 to 100% have been used for the validation. The method is found to be simple, rapid and more precise than other known techniques like IR or titration for %DDA measurements. The precision, ruggedness, robustness, specificity, stability and accuracy of the technique are discussed in this paper.

Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions

Abstract In this study we demonstrate that chitosan solutions can be neutralised up to physiological pH (∼7.2) using β-glycerol phosphate without creating immediate gel-like precipitation and furthermore that subsequent heating of these solutions induces hydrogel formation. The addition of the particular basic salt, glycerol phosphate, provides the correct buffering and other physicochemical conditions including control of hydrophobic interactions and hydrogen bonding which are necessary to retain chitosan in solution at neutral pH near 4°C and furthermore to allow gel formation upon heating to 37°C. Rheological investigation evidenced the endothermic gelation of chitosan/β-glycerol phosphate solutions and allowed the establishment of a sol/gel diagram. The gelation process appears to be governed by delicate interplay between the pH and the temperature. The role of β-glycerol phosphate is discussed in the light of relevant literature particularly those indicating the role of glycerol and polyols in the stabilisation of proteins and polysaccharides.

Optical and mechanical determination of poisson's ratio of adult bovine humeral articular cartilage

The equilibrium stiffness of articular cartilage is controlled by flow-independent elastic properties (Youngs modulus, ES, and Poissons ratio, v(s)) of the hydrated tissue matrix. In the current study, an optical (microscopic) method has been developed for the visualization of boundaries of cylindrical bovine humeral head articular cartilage disks (n = 9), immersed in physiological solution, and compressed in unconfined geometry. This method allowed a direct, model-independent estimation of Poissons ratio of the tissue at equilibrium, as well as characterization of the shape changes of the sample during the nonequilibrium dynamic phase. In addition to optical analyses, the equilibrium behavior of cartilage disks in unconfined and confined ramp-stress relaxation tests provided a direct estimation of the aggregate modulus, H(a) and Youngs modulus and, indirectly, Poissons ratio for the articular cartilage. The mean value for Poissons ratio obtained from the optical analysis was 0.185 +/- 0.065 (mean +/- S.D., n = 9). Values of elastic parameters obtained from the mechanical tests were 0.754 +/- 0.198 MPa, 0.677 +/- 0.223 MPa, and 0.174 +/- 0.106 for H(a), ES, and v(s), respectively (mean +/- S.D., n = 7). The similar v(s)-values obtained with optical and mechanical techniques imply that, at equilibrium for these two tests, the isotropic model is acceptable for mechanical analysis. However, the microscopic technique revealed that the lateral expansion, especially during the initial phase of relaxation, was inhomogeneous through the tissue depth. The superficial cartilage zone expanded less than the radial zone. The zonal differences in expansion were attributed to the known zonal differences in the fibrillar collagen architecture and proteoglycan concentration.

A molecular model of proteoglycan-associated electrostatic forces in cartilage mechanics

Measured values of the swelling pressure of charged proteoglycans (PG) in solution (Williams RPW, and Comper WD; Biophysical Chemistry 36:223, 1990) and the ionic strength dependence of the equilibrium modulus of PG-rich articular cartilage (Eisenberg SR, and Grodzinsky AJ; J Orthop Res 3: 148, 1985) are compared to the predictions of two models. Each model is a representation of electrostatic forces arising from charge present on spatially fixed macromolecules and spatially mobile micro-ions. The first is a macroscopic continuum model based on Donnan equilibrium that includes no molecular-level structure and assumes that the electrical potential is spatially invariant within the polyelectrolyte medium (i.e. zero electric field). The second model is based on a microstructural, molecular-level solution of the Poisson-Boltzmann (PB) equation within a unit cell containing a charged glycosaminoglycan (GAG) molecule and its surrounding atmosphere of mobile ions. This latter approach accounts for the space-varying electrical potential and electrical field between the GAG constituents of the PG. In computations involving no adjustable parameters, the PB-cell model agrees with the measured pressure of PG solutions to within experimental error (10%), whereas the ideal Donnan model overestimates the pressure by up to 3-fold. In computations involving one adjustable parameter for each model, the PB-cell model predicts the ionic strength dependence of the equilibrium modulus of articular cartilage. Near physiological ionic strength, the Donnan model overpredicts the modulus data by 2-fold, but the two models coincide for low ionic strengths (C0 < 0.025M) where the spatially invariant Donnan potential is a closer approximation to the PB potential distribution. The PB-cell model result indicates that electrostatic forces between adjacent GAGs predominate in determining the swelling pressure of PG in the concentration range found in articular cartilage (20-80 mg/ml). The PB-cell model is also consistent with data (Eisenberg and Grodzinsky, 1985, Lai WM, Hou JS, and Mow VC; J Biomech Eng 113: 245, 1991) showing that these electrostatic forces account for approximately 1/2 (290kPa) the equilibrium modulus of cartilage at physiological ionic strength while absolute swelling pressures may be as low as approximately 25-100kPa. This important property of electrostatic repulsion between GAGs that are highly charged but spaced a few Debye lengths apart allows cartilage to resist compression (high modulus) without generating excessive intratissue swelling pressures.

Chitosan-Glycerol Phosphate/Blood Implants Improve Hyaline Cartilage Repair in Ovine Microfracture Defects

BACKGROUND Microfracture is a surgical procedure that is used to treat focal articular cartilage defects. Although joint function improves following microfracture, the procedure elicits incomplete repair. As blood clot formation in the microfracture defect is an essential initiating event in microfracture therapy, we hypothesized that the repair would be improved if the microfracture defect were filled with a blood clot that was stabilized by the incorporation of a thrombogenic and adhesive polymer, specifically, chitosan. The objectives of the present study were to evaluate (1) blood clot adhesion in fresh microfracture defects and (2) the quality of the repair, at six months postoperatively, of microfracture defects that had been treated with or without chitosan-glycerol phosphate/blood clot implants, using a sheep model. METHODS In eighteen sheep, two 1-cm2 full-thickness chondral defects were created in the distal part of the femur and treated with microfracture; one defect was made in the medial femoral condyle, and the other defect was made in the trochlea. In four sheep, microfracture defects were created bilaterally; the microfracture defects in one knee received no further treatment, and the microfracture defects in the contralateral knee were filled with chitosan-glycerol phosphate/autologous whole blood and the implants were allowed to solidify. Fresh defects in these four sheep were collected at one hour postoperatively to compare the retention of the chitosan-glycerol phosphate/blood clot with that of the normal clot and to define the histologic characteristics of these fresh defects. In the other fourteen sheep, microfracture defects were made in only one knee and either were left untreated (control group; six sheep) or were treated with chitosan-glycerol phosphate/blood implant (treatment group; eight sheep), and the quality of repair was assessed histologically, histomorphometrically, and biochemically at six months postoperatively. RESULTS In the defects that were examined one hour postoperatively, chitosan-glycerol phosphate/blood clots showed increased adhesion to the walls of the defects as compared with the blood clots in the untreated microfracture defects. After histological processing, all blood clots in the control microfracture defects had been lost, whereas chitosanglycerol phosphate/blood clot adhered to and was partly retained on the surfaces of the defect. At six months, defects that had been treated with chitosan-glycerol phosphate/blood were filled with significantly more hyaline repair tissue (p < 0.05) compared with control defects. Repair tissue from medial femoral condyle defects that had been treated with chitosan-glycerol phosphate/blood contained more cells and more collagen compared with control defects and showed complete restoration of glycosaminoglycan levels. CONCLUSIONS Solidification of a chitosan-glycerol phosphate/blood implant in microfracture defects improved cartilage repair compared with microfracture alone by increasing the amount of tissue and improving its biochemical composition and cellular organization.

A Fibril-Network-Reinforced Biphasic Model of Cartilage in Unconfined Compression

Cartilage mechanical function relies on a composite structure of a collagen fibrillar network entrapping a proteoglycan matrix. Previous biphasic or poroelastic models of this tissue, which have approximated its composite structure using a homogeneous solid phase, have experienced difficulties in describing measured material responses. Progress to date in resolving these difficulties has demonstrated that a constitutive low that is successful for one test geometry (confined compression) is not necessarily successful for another (unconfined compression). In this study, we hypothesize that an alternative fibril-reinforced composite biphasic representation of cartilage can predict measured material responses and explore this hypothesis by developing and solving analytically a fibril-reinforced biphasic model for the case of uniaxial unconfined compression with frictionless compressing platens. The fibrils were considered to provide stiffness in tension only. The lateral stiffening provided by the fibril network dramatically increased the frequency dependence of disk rigidity in dynamic sinusoidal compression and the magnitude of the stress relaxation transient, in qualitative agreement with previously published data. Fitting newly obtained experimental stress relaxation data to the composite model allowed extraction of mechanical parameters from these tests, such as the rigidity of the fibril network, in addition to the elastic constants and the hydraulic permeability of the remaining matrix. Model calculations further highlight a potentially important difference between homogeneous and fibril-reinforced composite models. In the latter type of model, the stresses carried by different constituents can be dissimilar, even in sign (compression versus tension) even though strains can be identical. Such behavior, resulting only from a structurally physiological description, could have consequences in the efforts to understand the mechanical signals that determine cellular and extracellular biological responses to mechanical loads in cartilage.

Nonlinear analysis of cartilage in unconfined ramp compression using a fibril reinforced poroelastic model

OBJECTIVE To develop a biomechanical model for cartilage which is capable of capturing experimentally observed nonlinear behaviours of cartilage and to investigate effects of collagen fibril reinforcement in cartilage. DESIGN A sequence of 10 or 20 steps of ramp compression/relaxation applied to cartilage disks in uniaxial unconfined geometry is simulated for comparison with experimental data. BACKGROUND Mechanical behaviours of cartilage, such as the compression-offset dependent stiffening of the transient response and the strong relaxation component, have been previously difficult to describe using the biphasic model in unconfined compression. METHODS Cartilage is modelled as a fluid-saturated solid reinforced by an elastic fibrillar network. The latter, mainly representing collagen fibrils, is considered as a distinct constituent embedded in a biphasic component made up mainly of proteoglycan macromolecules and a fluid carrying mobile ions. The Youngs modulus of the fibrillar network is taken to vary linearly with its tensile strain but to be zero for compression. Numerical computations are carried out using a finite element procedure, for which the fibrillar network is discretized into a system of spring elements. RESULTS The nonlinear fibril reinforced poroelastic model is capable of describing the strong relaxation behaviour and compression-offset dependent stiffening of cartilage in unconfined compression. Computational results are also presented to demonstrate unique features of the model, e.g. the matrix stress in the radial direction is changed from tensile to compressive due to presence of distinct fibrils in the model. RELEVANCE Experimentally observed nonlinear behaviours of cartilage are successfully simulated, and the roles of collagen fibrils are distinguished by using the proposed model. Thus this study may lead to a better understanding of physiological responses of individual constituents of cartilage to external loads, and of the roles of mechanical loading in cartilage remodelling and pathology.

Vitrification of articular cartilage by high‐pressure freezing

For more than 20 years, high‐pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high‐pressure freezing. The cooling rates necessary to achieve this result under high‐pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed.

Drilling and microfracture lead to different bone structure and necrosis during bone-marrow stimulation for cartilage repair

Bone marrow stimulation is performed using several surgical techniques that have not been systematically compared or optimized for a desired cartilage repair outcome. In this study, we investigated acute osteochondral characteristics following microfracture and comparing to drilling in a mature rabbit model of cartilage repair. Microfracture holes were made to a depth of 2 mm and drill holes to either 2 mm or 6 mm under cooled irrigation. Animals were sacrificed 1 day postoperatively and subchondral bone assessed by histology and micro‐CT. We confirmed one hypothesis that microfracture produces fractured and compacted bone around holes, essentially sealing them off from viable bone marrow and potentially impeding repair. In contrast, drilling cleanly removed bone from the holes to provide access channels to marrow stroma. Our second hypothesis that drilling would cause greater osteocyte death than microfracture due to heat necrosis was not substantiated, because more empty osteocyte lacunae were associated with microfracture than drilling, probably due to shearing and crushing of adjacent bone. Drilling deeper to 6 mm versus 2 mm penetrated the epiphyseal scar in this model and led to greater subchondral hematoma. Our study revealed distinct differences between microfracture and drilling for acute subchondral bone structure and osteocyte necrosis. Additional ongoing studies suggest these differences significantly affect long‐term cartilage repair outcome.