Joseph W. Freeman

Collagen self-assembly and the development of tendon mechanical properties.

The development of the musculoskeleton and the ability to locomote requires controlled cell division as well as spatial control over deposition of extracellular matrix. Self-assembly of procollagen and its final processing into collagen fibrils occurs extracellularly. The formation of crosslinked collagen fibers results in the conversion of weak liquid-like embryonic tissues to tough elastic solids that can store energy and do work. Collagen fibers in the form of fascicles are the major structural units found in tendon. The purpose of this paper is to review the literature on collagen self-assembly and tendon development and to relate this information to the development of elastic energy storage in non-mineralizing and mineralizing tendons. Of particular interest is the mechanism by which energy is stored in tendons during locomotion. In vivo, collagen self-assembly occurs by the deposition of thin fibrils in recesses within the cell membrane. These thin fibrils later grow in length and width by lateral fusion of intermediates. In vitro, collagen self-assembly occurs by both linear and lateral growth steps with parallel events seen in vivo; however, in the absence of cellular control and enzymatic cleavage of the propeptides, the growth mechanism is altered, and the fibrils are irregular in cross section. Results of mechanical studies suggest that prior to locomotion the mechanical response of tendon to loading is dominated by the viscous sliding of collagen fibrils. In contrast, after birth when locomotion begins, the mechanical response is dominated by elastic stretching of crosslinked collagen molecules.

Viscoelastic properties of human skin and processed dermis.

Background/aims: The purpose of this work is to attempt to determine the elastic spring constant for collagen and elastic fibers (elastin) in skin and to detemine if the values of these elastic constants are similar to those reported for other tissues.

3D in vitro bioengineered tumors based on collagen I hydrogels

Cells cultured within a three-dimensional (3D) in vitro environment have the ability to acquire phenotypes and respond to stimuli analogous to in vivo biological systems. This approach has been utilized in tissue engineering and can also be applied to the development of a physiologically relevant in vitro tumor model. In this study, collagen I hydrogels cultured with MDA-MB-231 human breast cancer cells were bioengineered as a platform for in vitro solid tumor development. The cell-cell and cell-matrix interactions present during in vivo tissue progression were encouraged within the 3D hydrogel architecture, and the biocompatibility of collagen I supported unconfined cellular proliferation. The development of necrosis beyond a depth of ~150-200 μm and the expression of hypoxia-inducible factor (HIF)-1α were demonstrated in the in vitro bioengineered tumors. Oxygen and nutrient diffusion limitations through the collagen I matrix as well as competition for available nutrients resulted in growing levels of intra-cellular hypoxia, quantified by a statistically significant (p < 0.01) upregulation of HIF-1α gene expression. The bioengineered tumors also demonstrated promising angiogenic potential with a statistically significant (p < 0.001) upregulation of vascular endothelial growth factor (VEGF)-A gene expression. In addition, comparable gene expression analysis demonstrated a statistically significant increase of HIF-1α (p < 0.05) and VEGF-A (p < 0.001) by MDA-MB-231 cells cultured in the 3D collagen I hydrogels compared to cells cultured in a monolayer on two-dimensional tissue culture polystyrene. The results presented in this study demonstrate the capacity of collagen I hydrogels to facilitate the development of 3D in vitro bioengineered tumors that are representative of the pre-vascularized stages of in vivo solid tumor progression.

Collagen as a scaffold for biomimetic mineralization of vertebrate tissues

Collagen is a well known protein component that has the capacity to mineralize in a variety of vertebrate tissues. In its mineralized form, collagen potentially can be utilized as a biomimetic material for a variety of applications, including, for example, the augmentation and repair of damaged, congenitally defective, diseased or otherwise impaired calcified tissues such as bone and cartilage. In order to effect an optimal response in this regard, the manner in which collagen becomes mineralized is critically important to understand. This paper provides details concerning collagen–mineral interaction and its implications with respect to designing biomimetic mineralizing collagen that will be functionally competent in its biological, chemical, and biomechanical properties.

Coaxial electrospun poly(ε-caprolactone), multiwalled carbon nanotubes, and polyacrylic acid/polyvinyl alcohol scaffold for skeletal muscle tissue engineering†

Skeletal muscle repair after injury usually results in scar tissue and decreased functionality. In this study, we coaxially electrospun poly(ε-caprolactone), multiwalled carbon nanotubes, and a hydrogel consisting of polyvinyl alcohol and polyacrylic acid (PCL-MWCNT-H) to create a self-contained nanoactuating scaffold for skeletal muscle tissue replacement. This was then compared to electrospun PCL and PCL-MWCNT scaffolds. All scaffolds displayed some conductivity; however, MWCNT incorporation increased the conductivity. Only the PCL-MWCNT-H actuated when stimulated with 15 and 20 V. The PCL, PCL-MWCNT, and hydrogel only scaffolds demonstrated no reaction when 5, 8, 10, 15, and 20 V were applied. Thus, all components of the PCL-MWCNT-H scaffold are essential for movement. All three PCL-containing scaffolds were biocompatible, but the PCL-MWCNT-H scaffolds displayed more multinucleated cells with actin interaction. After tensile testing, the MWCNT-containing scaffolds had higher strength than the rat and pig skeletal muscle. Although the mechanical properties were higher than muscle, the PCL-MWCNT-H scaffold shows promise as a potential bioartificial nanoactuator for skeletal muscle.

Characterization of electrospun poly(L‐lactide) and gold nanoparticle composite scaffolds for skeletal muscle tissue engineering

Traumatic injuries can interrupt muscle contraction by damaging the skeletal muscle and/or the peripheral nerves. The healing process results in scar tissue formation that impedes muscle function. Electrospinning and metal nanoparticles (Nps) can create a scaffold that will trigger muscle cell elongation, orientation, fusion, and striation. Poly(L‐lactic acid) (PLLA) and gold (Au) Nps were electrospun to create three composite scaffolds, 7% Au–PLLA, 13% Au–PLLA and 21% Au–PLLA, and compared to PLLA alone. The scaffolds had a conductivity of 0.008 ± 0.003 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au–PLLA, 0.076 ± 0.004 S/cm for 13% Au–PLLA and 0.094 ± 0.037 S/cm for 21% Au–PLLA. Next, a cell study was conducted with rat primary muscle cells and all three Au–PLLA scaffolds. The first cell study showed low cell proliferation on all three of the Au–PLLA scaffolds; however, the second cell study showed that this was not due to Au Nps toxicity. Instead, low cell proliferation may be a marker for myotube differentiation and fusion. Values for the elastic modulus and yield stress for the Au–PLLA scaffolds on days 0, 7, 14, 21 and 28 were much higher than those for skeletal muscle tissue. Therefore, lower amounts of Au Nps may be utilized to create a biodegradable, biocompatible and conductive scaffold for skeletal muscle repair. Copyright

Evaluation of a hydrogel–fiber composite for ACL tissue engineering

The anterior cruciate ligament (ACL) is necessary for normal knee stability and movement. Unfortunately the ACL is also the most frequently injured ligament of the knee with severe disruptions requiring surgical intervention. In response to this, tissue engineering has emerged as an option for ACL replacement and repair. In this study we present a novel hydrogel-fibrous scaffold as a potential option for ACL replacement. The scaffold was composed of PLLA fibers, in a previously evaluated braid-twist structure, combined with a polyethylene glycol diacrylate (PEGDA) hydrogel to improve viscoelastic properties. Both hydrogel concentration (10%, 15%, and 20%) and amount of hydrogel (soaking the fibrous scaffold in hydrogel solution or encasing the scaffold in a block of hydrogel) were evaluated. It was found that the braid-twist scaffold had a greater porosity and larger number of pores above 100 μm than braided scaffolds with the same braiding angle. After testing for their effects on swelling, fiber degradation, and protein release, as well as viscoelastic and tensile testing (when combined with fibrous scaffolds), it was found that the composite scaffold soaked in 10% hydrogel had the best chemical release and mechanical properties. The optimized structure behaved similarly to natural ligament in tension with the addition of the hydrogel decreasing the ultimate tensile stress (UTS), but the UTS was still comparable to natural ACL. In addition, cellular studies showed that the hydrogel-PLLA fiber composite supported fibroblast growth.

Fabrication and mechanical characterization of 3D electrospun scaffolds for tissue engineering

Electrospinning is a polymer processing technique that produces fibrous structures comparable to the extracellular matrix of many tissues. Electrospinning, however, has been severely limited in its tissue engineering capabilities because this technique has produced few three-dimensional structures. Sintering of electrospun materials provides a method to fabricate unique architectures and allow much larger structures to be made. Electrospun mats were sintered into strips and cylinders, and their tensile and compressive mechanical properties were measured. In addition, electrospun materials with salt pores (salt embedded within the material and then leached out) were fabricated to improve porosity of the electrospun materials for tissue engineering scaffolds. Sintered electrospun poly(D,L-lactide) and poly(L-lactide) (PDLA/PLLA) materials have higher tensile mechanical properties (modulus: 72.3 MPa, yield: 960 kPa) compared to unsintered PLLA (modulus: 40.36 MPa, yield: 675.5 kPa). Electrospun PDLA/PLLA cylinders with and without salt-leached pores had compressive moduli of 6.69 and 26.86 MPa, respectively, and compressive yields of 1.36 and 0.56 MPa, respectively. Sintering of electrospun materials is a novel technique that improves electrospinning application in tissue engineering by increasing the size and types of electrospun structures that can be fabricated.

Recent Advancements in Ligament Tissue Engineering: The Use of Various Techniques and Materials for ACL Repair

The anterior cruciate ligament (ACL) is important for knee stability and kinematics. Unfortunately, it is also the most commonly injured ligament of the knee and due to its poor healing potential, severe damage warrants surgical intervention including complete replacement. Current ACL replacements have a host of limitations that prevent their extensive use. Thus, investigators have begun to pursue tissue engineering techniques to create new options for the repair, regeneration, and replacement of the ACL. These options involve devices that are mechanically functional tissue engineered scaffolds and as such are designed to withstand normal mechanical loads while promoting ligament development. This article presents background on the ACL and its replacement, novel replacement approaches utilizing natural polymers, synthetic polymers, and natural tissues, and recent patent coverage.

A link between stent radial forces and vascular wall remodeling: The discovery of an optimal stent radial force for minimal vessel restenosis

Coronary and peripheral artery disease (PAD) continue to be primary causes of morbidity and mortality in western nations; percutaneous transluminal angioplasty (PTA) with stenting has become a popular treatment. Unfortunately, restenosis is a significant problem following intravascular stent placement. This study considers the contribution of stent forces in vascular stenosis and remodeling to develop an equation for identifying the optimal stent force. z-Type stents of three radial forces [low (3.4 N), high (16.4 N), and ultrahigh (19.4 N)] were deployed into the iliac arteries of a juvenile porcine model. Vessel diameters were measured before, after deployment, and again at 30 days. At 30 days animals were killed and the vessels fixed in situ. After implantation, there was a significant increase in total thickness and neointimal hyperplasia with increasing stent force. The model for vessel radius and experimental data was in agreement. The model shows that maximum late‐term radius is achieved with a stent deployment stress of 480 kPa, which occurs at the end of the stress–strain curve nonlinear domain and beginning of the high-strain collagen domain. The results and calculations suggest that an optimal stent force exists that is subject to the geometry, structure, and mechanics of the target vessel. To achieve maximum late-term dilatation, stents should not produce stress in the vessel wall greater than the end of the transitional domain of the vessels stress–strain curve. This finding is extremely important for vascular stent development and will be expanded to preliminary vessel wall injury and atherosclerotic models.