Anowarul Islam

Collagen Substrate Stiffness Anisotropy Affects Cellular Elongation, Nuclear Shape, and Stem Cell Fate toward Anisotropic Tissue Lineage.

Rigidity of substrates plays an important role in stem cell fate. Studies are commonly carried out on isotropically stiff substrate or substrates with unidirectional stiffness gradients. However, many native tissues are anisotropically stiff and it is unknown whether controlled presentation of stiff and compliant material axes on the same substrate governs cytoskeletal and nuclear morphology, as well as stem cell differentiation. In this study, electrocompacted collagen sheets are stretched to varying degrees to tune the stiffness anisotropy (SA) in the range of 1 to 8, resulting in stiff and compliant material axes orthogonal to each other. The cytoskeletal aspect ratio increased with increasing SA by about fourfold. Such elongation was absent on cellulose acetate replicas of aligned collagen surfaces indicating that the elongation was not driven by surface topography. Mesenchymal stem cells (MSCs) seeded on varying anisotropy sheets displayed a dose-dependent upregulation of tendon-related markers such as Mohawk and Scleraxis. After 21 d of culture, highly anisotropic sheets induced greater levels of production of type-I, type-III collagen, and thrombospondin-4. Therefore, SA has direct effects on MSC differentiation. These findings may also have ramifications of stem cell fate on other anisotropically stiff tissues, such as skeletal/cardiac muscles, ligaments, and bone.

Fabrication of compositionally and topographically complex robust tissue forms by 3D-electrochemical compaction of collagen

Collagen solutions are phase-transformed to mechanically robust shell structures with curviplanar topographies using electrochemically-induced pH gradients. The process enables rapid layer-by-layer deposition of collagen-rich mixtures over the entire field simultaneously to obtain compositionally diverse multilayered structures. The in-plane tensile strength and modulus of the electrocompacted collagen sheet samples were 5200-fold and 2300-fold greater than those of the uncompacted collagen samples. Out-of-plane compression tests showed a 27-fold increase in compressive stress and a 46-fold increase in compressive modulus compared to uncompacted collagen sheets. Cells proliferated 4.9 times faster, and the cellular area spread was 2.7 times greater on compacted collagen sheets. Electrocompaction also resulted in a 2.9 times greater focal adhesion area than on regular collagen hydrogel. The reported improvements in the cell-matrix interactions with electrocompaction would serve to expedite the population of electrocompacted collagen scaffolds by cells. The capacity of the method to fabricate nonlinear curved topographies with compositional heterogeneous layers is demonstrated by sequential deposition of a collagen-hydroxyapatite layer over a collagen layer. The complex curved topography of the nasal structure is replicated by the electrochemical compaction method. The presented electrochemical compaction process is an enabling modality which holds significant promise for reconstruction of a wide spectrum of topographically complex systems such as joint surfaces, craniofacial defects, ears, nose, and urogenital forms.

Biomechanical evaluation of a novel suturing scheme for grafting load-bearing collagen scaffolds for rotator cuff repair

BACKGROUND Currently, there are no well-established suture protocols to attach fully load-bearing scaffolds which span tendon defects between bone and muscle for repair of critical sized tendon tears. Methods to attach load-bearing tissue repair scaffolds could enable functional repair of tendon injuries. METHODS Sixteen rabbit shoulders were dissected (New Zealand white rabbits, 1yr. old, female) to isolate the humeral-infraspinatus muscle complex. A unique suture technique was developed to allow for a 5mm segmental defect in infraspinatus tendon to be replaced with a mechanically strong bioscaffold woven from pure collagen threads. The suturing pattern resulted in a fully load-bearing scaffold. The tensile stiffness and strength of scaffold repair were compared with intact infraspinatus and regular direct repair. FINDINGS The failure load and displacement at failure of the scaffold repair group were 59.9N (standard deviation, SD=10.7) and 10.3mm (SD=2.9), respectively and matched those obtained by direct repair group which were 57.5N (SD=15.3) and 8.6mm (SD=1.5), (p>0.05). Failure load, displacement at failure and stiffness of both of the repair groups were half of the intact infraspinatus shoulder group. INTERPRETATION With the developed suture technique, scaffold repair showed similar failure load, displacement at failure and stiffness to the direct repair. This novel suturing pattern and the mechanical robustness of the scaffold at time zero indicates that the proposed model is mechanically viable for future in vivo studies which has a higher potential to translate into clinical uses.

Gamma Radiation Sterilization Reduces the High-cycle Fatigue Life of Allograft Bone.

BackgroundSterilization by gamma radiation impairs the mechanical properties of bone allografts. Previous work related to radiation-induced embrittlement of bone tissue has been limited mostly to monotonic testing which does not necessarily predict the high-cycle fatigue life of allografts in vivo.Questions/PurposesWe designed a custom rotating-bending fatigue device to answer the following questions: (1) Does gamma radiation sterilization affect the high-cycle fatigue behavior of cortical bone; and (2) how does the fatigue life change with cyclic stress level?MethodsThe high-cycle fatigue behavior of human cortical bone specimens was examined at stress levels related to physiologic levels using a custom-designed rotating-bending fatigue device. Test specimens were distributed among two treatment groups (n = 6/group); control and irradiated. Samples were tested until failure at stress levels of 25, 35, and 45 MPa.ResultsAt 25 MPa, 83% of control samples survived 30 million cycles (run-out) whereas 83% of irradiated samples survived only 0.5 million cycles. At 35 MPa, irradiated samples showed an approximately 19-fold reduction in fatigue life compared with control samples (12.2 × 106 ± 12.3 × 106 versus 6.38 × 105 ± 6.81 × 105; p = 0.046), and in the case of 45 MPa, this reduction was approximately 17.5-fold (7.31 × 105 ± 6.39 × 105 versus 4.17 × 104 ± 1.91 × 104; p = 0.025). Equations to estimate high-cycle fatigue life of irradiated and control cortical bone allograft at a certain stress level were derived.ConclusionsGamma radiation sterilization severely impairs the high cycle fatigue life of structural allograft bone tissues, more so than the decline that has been reported for monotonic mechanical properties. Therefore, clinicians need to be conservative in the expectation of the fatigue life of structural allograft bone tissues. Methods to preserve the fatigue strength of nonirradiated allograft bone tissue are needed.Clinical relevanceAs opposed to what monotonic tests might suggest, the cyclic fatigue life of radiation-sterilized structural allografts is likely severely compromised relative to the nonirradiated condition and therefore should be taken into consideration. Methods to reduce the effect of irradiation or to recover structural allograft bone tissue fatigue strength are important to pursue.

Effects of substrate stiffness on the tenoinduction of human mesenchymal stem cells

Extracellular matrix modulus plays an important role in regulating cell morphology, proliferation and differentiation during regular and diseased states. Although the effects of substrate topography and modulus on MSC differentiation are well known with respect to osteogenesis and adipogenesis, there has been relatively little investigation on the effects of this phenomenon on tenogenesis. Furthermore, relative roles of topographical factors (matrix alignment vs. matrix modulus) in inducing tenogenic differentiation is not well understood. In this study we investigated the effects of modulus and topographical alignment of type I collagen substrate on tendon differentiation. Type I collagen sheet substrates with random topographical alignment were fabricated with their moduli tuned in the range of 0.1, 1, 10 and 100MPa by using electrocompaction and controlled crosslinking. In one of the groups, topographical alignment was introduced at 10MPa stiffness, by controlled unidirectional stretching of the sheet. RT-PCR, immunohistochemistry and immunofluorescence results showed that mimicking the tendon topography, i.e. increasing the substrate modulus as well as alignment increased the tenogenic differentiation. Higher substrate modulus increased the expression of COLI, COLIII, COMP and TSP-4 about 2-3-fold and increased the production of COLI, COLIII and TSP-4 about 2-4-fold. Substrate alignment up regulated COLIII and COMP expression by 2-fold. Therefore, the tenoinductive collagen material model developed in this study can be used in the research and development of tissue engineering tendon repair constructs in future. STATEMENT OF SIGNIFICANCE Although the effects of substrate topography and modulus on MSC differentiation are well known with respect to osteogenesis and adipogenesis, there has been relatively little investigation on the effects of this phenomenon on tenogenesis. Furthermore, a relative role of topographical factors (matrix alignment vs. matrix modulus) in inducing tenogenic differentiation is not well understood. We investigated the effects of modulus and topographical alignment of type I collagen substrate on tendon differentiation. This study showed mimicking the tendon topography, i.e. increasing the substrate modulus as well as alignment increased the tenogenic differentiation. Therefore, the tenoinductive collagen material model developed in this study can be used in the research and development of tissue engineering tendon repair constructs in future.

Computer aided biomanufacturing of mechanically robust pure collagen meshes with controlled macroporosity.

Reconciliation of high strength and high porosity in pure collagen based structures is a major barrier in collagens use in load-bearing applications. The current study developed a CAD/CAM based electrocompaction method to manufacture highly porous patterned scaffolds using pure collagen. Utilization of computerized scaffold design and fabrication allows the integration of mesh-scaffolds with controlled pore size, shape and spacing. Mechanical properties of fabricated collagen meshes were investigated as a function of number of patterned layers, and with different pore geometries. The tensile stiffness, tensile strength and modulus ranges from 10-50 N cm(-1), 1-6 MPa and 5-40 MPa respectively for all the scaffold groups. These results are within the range of practical usability of different tissue engineering application such as tendon, hernia, stress urinary incontinence or thoracic wall reconstruction. Moreover, 3-fold increase in the layer number resulted in more than 5-fold increases in failure load, toughness and stiffness which suggests that by changing the number of layers and shape of the structure, mechanical properties can be modulated for the aforementioned tissue engineering application. These patterned scaffolds offer a porosity ranging from 0.8 to 1.5 mm in size, a range that is commensurate with pore sizes of repair meshes in the market. The connected macroporosity of the scaffolds facilitated cell-seeding such that cells populated the entire scaffold at the time of seeding. After 3 d of culture, cell nuclei became elongated. These results indicate that the patterned electrochemical deposition method in this study was able to develop mechanically robust, highly porous collagen scaffolds with controlled porosity which not only tries to solve one of the major tissue engineering problems at a fundamental level but also has a significant potential to be used in different tissue engineering applications.

Effects of PDGF-BB delivery from heparinized collagen sutures on the healing of lacerated chicken flexor tendon in vivo

Flexor tendon lacerations are traditionally repaired by using non-absorbable monofilament sutures. Recent investigations have explored to improve the healing process by growth factor delivery from the sutures. However, it is difficult to conjugate growth factors to nylon or other synthetic sutures. This study explores the performance of a novel electrochemically aligned collagen suture in a flexor tendon repair model with and without platelet derived growth factor following complete tendon laceration in vivo. Collagen suture was fabricated via electrochemical alignment process. Heparin was covalently bound to electrochemically aligned collagen sutures (ELAS) to facilitate affinity bound delivery of platelet-derived growth factor-BB (PDGF-BB). Complete laceration of the flexor digitorum profundus in the third digit of the foot was performed in 36 skeletally mature White Leghorn chickens. The left foot was used as the positive control. Animals were randomly divided into three groups: control specimens treated with standard nylon suture (n=12), specimens repaired with heparinated ELAS suture without PDGF-BB (n=12) and specimens repaired with heparinated ELAS suture with affinity bound PDGF-BB (n=12). Specimens were harvested at either 4weeks or 12weeks following tendon repair. Differences between groups were evaluated by the degree of gross tendon excursion, failure load/stress, stiffness/modulus, absorbed energy at failure, elongation/strain at failure. Quantitative histological scoring was performed to assess cellularity and vascularity. Closed flexion angle measurements demonstrated no significant differences in tendon excursion between the study groups at 4 or 12weeks. Biomechanical testing showed that the group treated with PDGF-BB bound heparinated ELAS suture had significantly higher stiffness and failure load (p<0.05) at 12-weeks relative to both heparinated ELAS suture and nylon suture. Similarly, the group treated with PDGF-BB bound suture had significantly higher ultimate tensile strength and Youngs modulus (p<0.05) at 12-weeks relative to both ELAS suture and nylon suture. Compared to nylon controls, heparinized ELAS with PDGF-BB improved biomechanics and vascularity during tendon healing by 12-weeks following primary repair. The ability of ELAS to deliver PDGF-BB to the lacerated area of tendon presents investigators with a functional bioinductive platform to improve repair outcomes following flexor tendon repair. STATEMENT OF SIGNIFICANCE A high strength aligned collagen suture was fabricated via linear electrocompaction and heparinized for prolonged delivery of PDFG-BB. When it was used to suture a complete lacerated flexor tendon in a chicken model controlled release of the PDGF-BB improved the strength of treated tendon after 12 weeks compared to tendon sutured with commercial nylon suture. Furthermore, Collagen suture with affinity bound PDGF-BB enhanced the vascularization and remodeling of lacerated tendon when it compare to synthetic nylon suture. Overall, electrocompacted collagen sutures holds potential to improve repair outcome in flexor tendon surgeries by improving repair strength and stiffness, vascularity, and remodeling via sustained delivery of the PDGF-BB. The bioinductive collagen suture introduces a platform for sustained delivery of other growth factors for a wide-array of applications.