Investigating Cancerous Exosomes' Effects on CD8+ T-Cell IL-2 Production in a 3D Unidirectional Flow Bioreactor Using 3D Printed, RGD-Functionalized PLLA Scaffolds.

Exosomes from cancer cells are implicated in cancer progression and metastasis, carrying immunosuppressive factors that limit the antitumor abilities of immune cells. The development of a real-time, 3D cell/scaffold construct flow perfusion system has been explored as a novel tool in the study of T-cells and exosomes from cancer cells. Exosomes from human lung cancer (H1299 and A549) cells were co-cultured in a unidirectional flow bioreactor with CD8+ T-cells immobilized onto 3D-printed RGD-functionalized poly(L-lactic) acid (PLLA) scaffolds and assessed for IL-2 production. The IL-2 production was investigated for a wide range of T-cell to exosome ratios. With the successful incorporation of the RGD binding motif onto the PLLA surface at controllable densities, CD8+ T-cells were successfully attached onto 2D disks and 3D printed porous PLLA scaffolds. T-cell attachment increased with increasing RGD surface density. The diameter of the attached T-cells was 7.2 ± 0.2 µm for RGD densities below 0.5 nmoles/mm2 but dropped to 5.1 ± 0.3 µm when the RGD density was 2 nmoles/mm2 due to overcrowding. The higher the number of cancer exosomes, the less the IL-2 production by the surface-attached T-cells. In 2D disks, the IL-2 production was silenced for T-cell to exosome ratios higher than 1:10 in static conditions. IL-2 production silencing in static 3D porous scaffolds required ratios higher than 1:20. The incorporation of flow resulted in moderate to significant T-cell detachment. The portions of T-cells retained on the 3D scaffolds after exposure for 4 h to 0.15 or 1.5 mL/min of perfusion flow were 89 ± 11% and 30 ± 8%, respectively. On 3D scaffolds and in the presence of flow at 0.15 ml/min, both H1299 and A549 cancerous exosomes significantly suppressed IL-2 production for T-cell to exosome ratios of 1:1000. The much higher level of exosomes needed to silence the IL-2 production from T-cells cultured under unidirectional flow, compared to static conditions, denotes the importance of the culturing conditions and the hydrodynamic environment, on the interactions between CD8+ T-cells and cancer exosomes.

Tuning the biomimetic behavior of scaffolds for regenerative medicine through surface modifications.

Tissue engineering and regenerative medicine rely extensively on biomaterial scaffolds to support cell adhesion, proliferation, and differentiation physically and chemically in vitro and in vivo. Changes to the surface characteristics of the scaffolds have the greatest impact on cell response. Here, we discuss five dominant surface modification approaches used to biomimetically improve the most common scaffolds for tissue engineering, those based on aliphatic polyesters. Scaffolds of aliphatic polyesters such as poly(l-lactic acid), poly(l-lactic-co-glycolic acid), and poly(ε-caprolactone) are often used in tissue engineering because they provide desirable, tunable properties such as ease of manufacturing, good mechanical properties, and nontoxic degradation products. However, cell-surface interactions necessary for tissue engineering are limited on these materials by their smooth postfabrication surfaces, hydrophobicity, and lack of recognizable biochemical binding sites. The surface modification techniques that have been developed for synthetic polymer scaffolds reduce initial barriers to cell adhesion, proliferation, and differentiation. Topographical modification, protein adsorption, mineral coating, functional group incorporation, and biomacromolecule immobilization each contribute through varying mechanisms to improving cell interactions with aliphatic polyester scaffolds. Furthermore, rational combination of methods from these categories can provide nuanced, specific environments for targeted tissue development.

The effects of varying frequency and duration of mechanical stimulation on a tissue-engineered tendon construct.

Decellularized, discarded human tissues, such as the human umbilical vein, have been widely utilized for tissue engineering applications, including tendon grafts. When recellularized, such natural scaffolds are cultured in 3D dynamic culture environments (bioreactor systems). For tendon tissue-engineered grafts, such systems often employ oscillatory mechanical stimulation in the form uniaxial tensile strain. The three main parameters of such stimulation are frequency, duration, and force. In this study we investigated the effects of changing the duration (0.5, 1, and 2 h/day) and frequency (0.5, 1, 2 cycles/min) of stimulation of a human umbilical vein seeded with mesenchymal stem cells cultured for up to 7 days. Strain of the construct was held constant at 2%. The highest proliferation rates were observed in the 0.5 h/day duration and 1 cycle/min frequency (203% increase) with a close second being 1 h/day and 1 cycle/min frequency (170% increase). Static cultures along with a 2 cycles/min frequency and a 2 h/day duration of stretching did not increase cellular proliferation significantly. Extracellular matrix quality and alignment of the construct fibers had a direct relation to cellularity and those groups with the highest cellularity improved the most. Gene expression indicated cellular activity consistent with tendon-like tissue remodeling. In addition, scleraxis, tenascin-C, and tenomodulin were upregulated in certain groups after 7 days, with osteoblast, chondrocyte, and adipocyte phenotypes depressed. The stimulation parameters investigated in this study indicated that slower frequencies and shorter durations were best for construct quality in early stage cultures.

Effects of Shear Stress Gradients on Ewing Sarcoma Cells Using 3D Printed Scaffolds and Flow Perfusion.

In this work, we combined three-dimensional (3D) scaffolds with flow perfusion bioreactors to evaluate the gradient effects of scaffold architecture and mechanical stimulation, respectively, on tumor cell phenotype. As cancer biologists elucidate the relevance of 3D in vitro tumor models within the drug discovery pipeline, it has become more compelling to model the tumor microenvironment and its impact on tumor cells. In particular, permeability gradients within solid tumors are inherently complex and difficult to accurately model in vitro. However, 3D printing can be used to design scaffolds with complex architecture, and flow perfusion can simulate mechanical stimulation within the tumor microenvironment. By modeling these gradients in vitro with 3D printed scaffolds and flow perfusion, we can identify potential diffusional limitations of drug delivery within a tumor. Ewing sarcoma (ES), a pediatric bone tumor, is a suitable candidate to study heterogeneous tumor response due to its demonstrated shear stress-dependent secretion of ligands important for ES tumor progression. We cultured ES cells under flow perfusion conditions on poly(propylene fumarate) scaffolds, which were fabricated with a distinct pore size gradient via extrusion-based 3D printing. Computational fluid modeling confirmed the presence of a shear stress gradient within the scaffolds and estimated the average shear stress that ES cells experience within each layer. Subsequently, we observed enhanced cell proliferation under flow perfusion within layers supporting lower permeability and increased surface area. Additionally, the effects of shear stress gradients on ES cell signaling transduction of the insulin-like growth factor-1 pathway elicited a response dependent upon the scaffold gradient orientation and the presence of flow-derived shear stress. Our results highlight how 3D printed scaffolds, in combination with flow perfusion in vitro, can effectively model aspects of solid tumor heterogeneity for future drug testing and customized patient therapies.

Monitoring Bone Tissue Engineered (BTE) Constructs Based on the Shifting Metabolism of Differentiating Stem Cells.

Ever-increasing demand for bone grafts necessitates the realization of clinical implementation of bone tissue engineered constructs. The predominant hurdle to implementation remains to be securing FDA approval, based on the lack of viable methods for the rigorous monitoring of said constructs. The study presented herein details a method for such monitoring based on the shifting metabolism of mesenchymal stem cells (MSCs) as they differentiate into osteoblasts. To that end, rat MSCs seeded on 85% porous spunbonded poly(L-lactic acid) scaffolds were cultured in flow perfusion bioreactors with baseline or osteoinductive media, and levels of key physio-metabolic markers (oxygen, glucose, osteoprotegerin, and osteocalcin) were monitored throughout culture. Comparison of these non-destructively obtained values and current standard destructive analyses demonstrated key trends useful for the concurrent real-time monitoring of construct cellularity and maturation. Principle among these is the elucidation of the ratio of the rates of oxygen uptake to glucose consumption as a powerful quality marker. This ratio, supported on a physiological basis, has been shown herein to be reliable in the determination of both construct maturation (defined as osteoblastic differentiation and accompanying mineralization) and construct cellularity. Supplementary monitoring of OPG and OCN are shown to provide further validation of such metrics.

Antitumor Synergism and Enhanced Survival with a Tumor Vasculature-Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide.

Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug-treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855-65. ©2017 AACR.

Tenocytic extract and mechanical stimulation in a tissue-engineered tendon construct increases cellular proliferation and ECM deposition.

Chemical and mechanical stimulation, when properly utilized, positively influence both the differentiation of in vitro cultured stem cells and the quality of the deposited extracellular matrix (ECM). This study aimed to find if cell-free extract from primary tenocytes can positively affect the development of a tissue-engineered tendon construct, consisting of a human umbilical vein (HUV) seeded with mesenchymal stem cells (MSCs) subjected to cyclical mechanical stimulation. The tenocytic cell-free extract possesses biological material from tendon cells that could potentially influence MSC tenocytic differentiation and construct development. We demonstrate that the addition of tenocytic extract in statically cultured tendon constructs increases ECM deposition and tendon-related gene expression of MSCs. The incorporation of mechanical stimulation (2% strain for 30 min/day at 0.5 cycles/min) with tenocytic extract further improved the MSC seeded HUV constructs by increasing cellularity of the construct by 37% and the ultimate tensile strength by 33% compared to the constructs with only mechanical stimulation after 14 days. Furthermore, the addition of mechanical stimulation to the extract supplementation produced longitudinal ECM fibril alignment along with dense connective tissue, reminiscent of natural tendon.

Sensing metabolites for the monitoring of tissue engineered construct cellularity in perfusion bioreactors.

As the field of tissue engineering progresses ever-further toward realizing clinical implementation of tissue-engineered constructs for wound regeneration, perhaps the most significant hurdle remains the establishment of non-destructive means for real-time in vitro assessment. In order to address this barrier, the study presented herein established the viability of the development of correlations between metabolic rates (specifically oxygen uptake, glucose consumption, and lactate production) and the cellularity of tissue-engineered cultures comprised of rat mesenchymal stem cells dynamically seeded on 85% porous nonwoven spunbonded poly(l-lactic acid) fiber mesh scaffolds. Said scaffolds were cultured for up to 21 days in a flow perfusion bioreactor system wherein α-MEM (supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic) was perfused directly through each scaffold at low flow rates (~0.15mL/min). Metabolite measurements were obtained intermittently through the use of a fiber-optic probe (for the case of oxygen) and biochemical assays (for glucose and lactate). Such measurements were subsequently correlated with cellularity data obtained utilizing current-standard destructive means. The resulting correlations, all exhibiting high R2 values, serve as a proof-on-concept for the use of metabolic data for the determination of scaffold cellularity in real-time non-destructively. This study can be easily adapted for use with various cell types, media formulations, and potentially different bioreactor systems. Implementation of more advanced in situ measurement devices could be easily accommodated to allow for true real-time, on-line metabolite monitoring and cellularity estimation.

Antitumor activity of an enzyme prodrug therapy targeted to the breast tumor vasculature.

The L-methioninase-annexin V/selenomethionine enzyme prodrug system, designed to target the tumor vasculature and release the methylselenol anticancer drug in the tumor, was tested in mice with implanted MBA-MB-231 breast tumors. This therapy was able to cause a reduction in the size of the tumors during the treatment period. It was shown that L-methioninase-annexin V was uniformly bound at the blood vessel surface in the tumor and also that there was a substantial cutoff of blood flowing through the treated tumor, consistent with the therapy's design. This new approach for enzyme prodrug therapy of breast cancer appears promising.

Mechanical and in vitro investigation of a porous PEEK foam for medical device implants.

PURPOSE: Implantable-grade polyetheretherketone (PEEK-OPTIMA®) is a high-performance thermoplastic that has been used in implant devices such as spinal-fusion cages since its introduction in 1999. Here, a new porous PEEK version was investigated. METHODS: Porous PEEK was fabricated using industrial scale relevant methods of compounding with porogen filler, extrusion, and subsequent extraction with water at supercritical temperatures and pressures. Mechanical properties were assessed according to ISO standards. Marrow stromal cells were cultured on porous PEEK samples and in vitro cytocompatibility was assessed by total DNA, alkaline phosphatase activity, osteopontin, calcium, and cell morphology to indicate stages of proliferation, differentiation, and mineralization. Compressive strength was assessed statically on 21 day cell cultures and media-soaked samples and dynamically within a medical device application specific context for interbody fusion cages (ASTM F2077). RESULTS: Manufacturing resulted in a biomaterial with ~50% porosity and a mean pore size of 100 microns. The porous PEEK was found to have: tensile strength (14.5MPa), strain at break (3.5%), impact strength (3.6 kJ/m2), flexural strength (21.6MPa), and flexural modulus (0.8GPa). Production of extracellular mineralized matrix occurred very early in the culture period, indicating a preferred surface for differentiation. SEM images revealed polygonal cell morphology supporting a differentiated osteoblastic-like phenotype. EDS analysis detected levels of carbon, phosphorus, and calcium coinciding with assay results for the proliferation and differentiation stages. CONCLUSION: Previous observations of cytocompatibility and calcification on the PEEK biomaterial could be carried through to this new porous form of the PEEK biomaterial. This helps porous PEEK to potentially offer more design options for implant devices requiring reduced modulus and/or increased tissue ingrowth aspects at the surface.

3D tissue-engineered construct analysis via conventional high-resolution microcomputed tomography without X-ray contrast.

As the field of tissue engineering develops, researchers are faced with a large number of degrees of freedom regarding the choice of material, architecture, seeding, and culturing. To evaluate the effectiveness of a tissue-engineered strategy, histology is typically done by physically slicing and staining a construct (crude, time-consuming, and unreliable). However, due to recent advances in high-resolution biomedical imaging, microcomputed tomography (µCT) has arisen as a quick and effective way to evaluate samples, while preserving their structure in the original state. However, a major barrier for using µCT to do histology has been its inability to differentiate between materials with similar X-ray attenuation. Various contrasting strategies (hardware and chemical staining agents) have been proposed to address this problem, but at a cost of additional complexity and limited access. Instead, here we suggest a strategy for how virtual 3D histology in silico can be conducted using conventional µCT, and we provide an illustrative example from bone tissue engineering. The key to our methodology is an implementation of scaffold surface architecture that is ordered in relation to cells and tissue, in concert with straightforward image-processing techniques, to minimize the reliance on contrasting for material segmentation. In the case study reported, µCT was used to image and segment porous poly(lactic acid) nonwoven fiber mesh scaffolds that were seeded dynamically with mesenchymal stem cells and cultured to produce soft tissue and mineralized tissue in a flow perfusion bioreactor using an osteogenic medium. The methodology presented herein paves a new way for tissue engineers to identify and distinguish components of cell/tissue/scaffold constructs to easily and effectively evaluate the tissue-engineering strategies that generate them.

Predicting the stress distribution within scaffolds with ordered architecture.

Current tissue engineering technologies involve the seeding of cells on porous scaffolds, within which the cells can proliferate and differentiate, when cultured in bioreactors. The flow of culture media through the scaffolds generates stresses that are important for both cell differentiation and cell growth. A recent study [Appl. Phys. Lett. 97 (2010), 024101] showed that flow-induced stresses inside highly porous and randomly structured scaffolds follow a three-point gamma probability density function (p.d.f.). The goal of the present study is to further investigate whether the same p.d.f. can also describe the distribution of stresses in structured porous scaffolds, what is the range of scaffold porosity for which the distribution is valid, and what is the physical reason for such behavior. To do that, the p.d.f. of flow-induced stresses in different scaffold geometries were calculated via flow dynamics simulations. It was found that the direction of flow relative to the internal architecture of the scaffolds is important for stress distributions. The stress distributions follow a common distribution within statistically acceptable accuracy, when the flow direction does not coincide with the direction of internal structural elements of the scaffold.

Friction coefficients for mechanically damaged bovine articular cartilage.

We used a pin-on-disc tribometer to measure the friction coefficient of both pristine and mechanically damaged cartilage samples in the presence of different lubricant solutions. The experimental set up maximizes the lubrication mechanism due to interstitial fluid pressurization. In phosphate buffer solution (PBS), the measured friction coefficient increases with the level of damage. The main result is that when poly(ethylene oxide) (PEO) or hyaluronic acid (HA) are dissolved in PBS, or when synovial fluid (SF) is used as lubricant, the friction coefficients measured for damaged cartilage samples are only slightly larger than those obtained for pristine cartilage samples, indicating that the surface damage is in part alleviated by the presence of the various lubricants. Among the lubricants considered, 100 mg/mL of 100,000 Da MW PEO in PBS appears to be as effective as SF. We attempted to discriminate the lubrication mechanism enhanced by the various compounds. The lubricants viscosity was measured at shear rates comparable to those employed in the friction experiments, and a quartz crystal microbalance with dissipation monitoring was used to study the adsorption of PEO, HA, and SF components on collagen type II adlayers pre-formed on hydroxyapatite. Under the shear rates considered the viscosity of SF is slightly larger than that of PBS, but lower than that of lubricant formulations containing HA or PEO. Neither PEO nor HA showed strong adsorption on collagen adlayers, while evidence of adsorption was found for SF. Combined, these results suggest that synovial fluid is likely to enhance boundary lubrication. It is possible that all three formulations enhance lubrication via the interstitial fluid pressurization mechanism, maximized by the experimental set up adopted in our friction tests.

Long-term in vivo effect of PEG bone tissue engineering scaffolds.

Polyethylene glycol (PEG) performs multiple roles for bone tissue engineering scaffolds. Successful in vivo implantation for long periods of time requires a scaffold that is biocompatible, osteoconductive, osteoinductive, and promotes cell recruitment and attachment. PEG has significant advantages such as excellent biocompatibility and flexibility, but certain drawbacks such as poor mechanical strength and cell attachment limit its use as a plain scaffold. Instead, it is often used as an additive, composite, or delivery system. Below is a summary of current research involving the use of PEG-based biomaterials in bone tissue engineering, specifically with regard to long term in vivo effects.

The effect of cell seeding density on the cellular and mechanical properties of a mechanostimulated tissue-engineered tendon.

The initial seeding density is a critical variable in functional tissue engineering. A sufficient number of cells uniformly distributed throughout the scaffold is a key requirement to achieve homogeneous extracellular matrix deposition in vitro. However, high initial seeding densities might have negative repercussions on nutrient availability, cellular metabolism, and cell viability. In the current study, our aim was to understand the implications of using high seeding densities (3, 5, and 10 million cells/mL) in a human umbilical vein (HUV) tendon model subjected to 1 h of cyclic stretching per day at 2% strain and a frequency of 0.0167 Hz in a mechanostimulating bioreactor, on nutrient availability, cell viability and metabolism, and construct properties. Mechanostimulated constructs seeded with 3 million cells/mL had significantly higher cell number than the static controls and resulted in a 20-fold increase in proliferation rates and a 3-fold increase in tensile strength values after 1 week of culture in the bioreactor. However, higher seeding densities resulted in cell death, degraded extracellular matrix, and poorer mechanical properties. Nutrient and growth factor mass transport limitations are implicated in the inability of the decellularized HUV to support high cell numbers. The effective diffusion coefficient for glucose was measured to be 0.21±0.04 cm(2)/day. In the absence of convective flow, proteins and growth factors with a molecular radius larger than 4.9 nm could not diffuse through the HUV. Cells seeded in the HUV consumed 10.5±0.5 ng/cell/day of glucose. Glucose diffusion coefficient and glucose consumption rates in the HUV indicated the presence of glucose mass transport limitations when cell seeding densities exceed 3 million cells/mL.

Experimental friction coefficients for bovine cartilage measured with a pin-on-disk tribometer: testing configuration and lubricant effects.

The friction coefficient between wet articular cartilage surfaces was measured using a pin-on-disk tribometer adopting different testing configurations: cartilage-on-pin vs. alumina-on-disk (CA); cartilage-on-pin vs. cartilage-on-disk (CC); and alumina-on-pin vs. cartilage-on-disk (AC). Several substances were dissolved in the phosphate buffered saline (PBS) solution to act as lubricants: 10,000 molecular weight (MW) polyethylene glycol (PEG), 100,000 MW PEG, and chondroitin sulfate (CS), all at 100 mg/mL concentration. Scanning electron microscopy photographs of the cartilage specimens revealed limited wear due to the experiment. Conducting the experiments in PBS solutions we provide evidence according to which a commercial pin-on-disk tribometer allows us to assess different lubrication mechanisms active in cartilage. Specifically, we find that the measured friction coefficient strongly depends on the testing configuration. Our results show that the friction coefficient measured under CC and AC testing configurations remains very low as the sliding distance increases, probably because during the pin displacement the pores present in the cartilage replenish with PBS solution. Under such conditions the fluid phase supports a large load fraction for long times. By systematically altering the composition of the PBS solution we demonstrate the importance of solution viscosity in determining the measured friction coefficient. Although the friction coefficient remains low under the AC testing configuration in PBS, 100 mg/mL solutions of both CS and 100,000 MW PEG in PBS further reduce the friction coefficient by ~40%. Relating the measured friction coefficient to the Hersey number, our results are consistent with a Stribeck curve, confirming that the friction coefficient of cartilage under the AC testing configuration depends on a combination of hydrodynamic, boundary, and weep bearing lubrication mechanisms.

Enhanced osteoblastic differentiation of mesenchymal stem cells seeded in RGD-functionalized PLLA scaffolds and cultured in a flow perfusion bioreactor.

The present study combines chemical and mechanical stimuli to modulate the osteogenic differentiation of mesenchymal stem cells (MSCs). Arg-Gly-Asp (RGD) peptides incorporated into biomaterials have been shown to upregulate MSC osteoblastic differentiation. However, these effects have been assessed under static culture conditions, while it has been reported that flow perfusion also has an enhancing effect on MSC osteoblastic differentiation. It is clear that there is a need to combine RGD modification of biomaterials with mechanical stimulation of MSCs via flow perfusion and evaluate its effects on MSC differentiation down the osteogenic lineage. In this study, the effect of different levels of RGD modification of poly(L-lactic acid) scaffolds on MSC osteogenesis was evaluated under conditions of flow perfusion. It was found that there is a synergistic enhancement of different osteogenic markers, due to the combination of flow perfusion and RGD surface modification when compared to their individual effects. Furthermore, under conditions of flow perfusion, there is an RGD surface concentration optimal for differentiation, and it is flow rate-dependent. This report underlines the significance of incorporating combined biomimesis via biochemical and mechanical microenvironments that modulate in vivo cell behaviour and tissue function for more efficient tissue-engineering strategies.

Computational modeling of flow-induced shear stresses within 3D salt-leached porous scaffolds imaged via micro-CT.

Flow-induced shear stresses have been found to be a stimulatory factor in pre-osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of porous scaffolds, analytical estimation of the local shear forces is impractical. The primary goal of this work is to investigate the shear stress distributions within Poly(l-lactic acid) scaffolds via computation. Scaffolds used in this study are prepared via salt leeching with various geometric characteristics (80-95% porosity and 215-402.5microm average pore size). High resolution micro-computed tomography is used to obtain their 3D structure. Flow of osteogenic media through the scaffolds is modeled via lattice Boltzmann method. It is found that the surface stress distributions within the scaffolds are characterized by long tails to the right (a positive skewness). Their shape is not strongly dependent on the scaffold manufacturing parameters, but the magnitudes of the stresses are. Correlations are prepared for the estimation of the average surface shear stress experienced by the cells within the scaffolds and of the probability density function of the surface stresses. Though the manufacturing technique does not appear to affect the shape of the shear stress distributions, presence of manufacturing defects is found to be significant: defects create areas of high flow and high stress along their periphery. The results of this study are applicable to other polymer systems provided that they are manufactured by a similar salt leeching technique, while the imaging/modeling approach is applicable to all scaffolds relevant to tissue engineering.

Preparation of a functionally flexible, three-dimensional, biomimetic poly(L-lactic acid) scaffold with improved cell adhesion.

Poly(L-lactic acid) (PLLA) is widely used in tissue-engineering applications because of its degradation characteristics and mechanical properties, but it possesses an inert nature, affecting cell-matrix interactions. It is desirable to modify the surface of PLLA to create biomimetic scaffolds that will enhance tissue regeneration. We prepared a functionally flexible, biomimetic scaffold by derivatizing the surface of PLLA foams into primary amines, activated pyridylthiols, or sulfhydryl groups, allowing a wide variety of modifications. Poly(L-lysine) (polyK) was physically entrapped uniformly throughout the scaffold surface and in a controllable fashion by soaking the foams in an acetone-water mixture and later in a polyK solution in dimethylsulfoxide. Arginine-glycine-aspartic acid-cysteine (RGDC) adhesion peptide was linked to the polyK via creating disulfide bonds introduced through the use of the linker N-succinimidyl-3-(2-pyridylthiol)-propionate. Presence of RGDC on the surface of PLLA 2-dimensional (2-D) disks and 3-D scaffolds increased cell surface area and the number of adherent mesenchymal stem cells. We have proposed a methodology for creating biomimetic scaffolds that is easy to execute, flexible, and nondestructive.

Improved mesenchymal stem cell seeding on RGD-modified poly(L-lactic acid) scaffolds using flow perfusion.

Arg-Gly-Asp (RGD) has been widely utilized to increase cell adhesion to three-dimensional scaffolds for tissue engineering. However, cell seeding on these scaffolds has only been carried out statically, which yields low cell seeding efficiencies. We have characterized, for the first time, the seeding of rat mesenchymal stem cells on RGD-modified poly(L-lactic acid) (PLLA) foams using oscillatory flow perfusion. The incorporation of RGD on the PLLA foams improves scaffold cellularity in a dose-dependent manner under oscillatory flow perfusion seeding. When compared to static seeding, oscillatory flow perfusion is the most efficient seeding technique. Cell detachment studies show that cell adhesion is dependent on the applied flow rate, and that cell attachment is strengthened at higher levels of RGD modification.