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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/mm 2 but dropped to 5.1 ± 0.3 µm when the RGD density was 2 nmoles/mm 2 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.

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., (© 2019 John Wiley & Sons, Ltd.)

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.

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.

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.

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 ., (©2017 American Association for Cancer Research.)

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 R 2 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

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.

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 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., (Copyright © 2010 John Wiley & Sons, Ltd.)

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.

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.

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., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site., (Copyright 2007 Wiley Periodicals, Inc.)

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.

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.

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell-matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-microm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell-matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.

This study instituted a unique approach to bone tissue engineering by combining effects of mechanical stimulation in the form of fluid shear stresses and the presence of bone-like extracellular matrix (ECM) on osteodifferentiation. Rat marrow stromal cells (MSCs) harvested from bone marrow were cultured on titanium (Ti) fiber mesh discs for 12 days in a flow perfusion system to generate constructs containing bone-like ECM. To observe osteodifferentiation and bone-like matrix deposition, these decellularized constructs and plain Ti fiber meshes were seeded with MSCs (Ti/ECM and Ti, respectively) and cultured in the presence of fluid shear stresses either with or without the osteogenic culture supplement dexamethasone. The calcium content, alkaline phosphatase activity, and osteopontin secretion were monitored as indicators of MSC differentiation. Ti/ECM constructs demonstrated a 75-fold increase in calcium content compared with their Ti counterparts after 16 days of culture. After 16 days, the presence of dexamethasone enhanced the effects of fluid shear stress and the bone-like ECM by increasing mineralization 50-fold for Ti/ECM constructs; even in the absence of dexamethasone, the Ti/ECM constructs exhibited approximately a 40-fold increase in mineralization compared with their Ti counterparts. Additionally, denatured Ti/ECM* constructs demonstrated a 60-fold decrease in calcium content compared with Ti/ECM constructs after 4 days of culture. These results indicate that the inherent osteoinductive potential of bone-like ECM along with fluid shear stresses synergistically enhance the osteodifferentiation of MSCs with profound implications on bone-tissue-engineering applications.

Muskuloskeletal tissue includes bone, cartilage, ligament, skeletal muscle and tendons. These tissues malfunction either due to a natural injury, trauma, or a disorder. In all cases natural regeneration needs to be enhanced by medication and, in many instances, by surgery. Surgical techniques are limited to suturing, autografts or allografts. Tissue engineering stems from the challenge presented by the limited resources for natural implants and the ineffectiveness of previous curing techniques. The challenge in tissue engineering resides in the design of a functional bioreactor that would: (1) house the engineered construct under sterile conditions; and (2) provide the appropriate stimuli that would result in a neotissue with biochemical and biomechanical properties comparable to in situ tissue. The various types and designs of bioreactors for the regeneration of musculoskeletal tissue, including spinner flask, rotating wall vessel, flow perfusion, and mechanical loading devices are presented in this paper.

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 microm fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 x 10(5) cells in 300 microl, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.

The objective of this study was to evaluate the attachment, proliferation, and differentiation of rat mesenchymal stem cells (MSC) toward the osteoblastic phenotype seeded on polypyrrole (PPy) thin films made by admicellar polymerization. Three different concentrations of pyrrole (Py) monomer (20, 35, and 50 x 10(-3) M) were used with the PPy films deposited on tissue culture polystyrene dishes (TCP). Regular TCP dishes and PPy polymerized on TCP by chemical polymerization without surfactant using 5 x 10(-3) M Py, were used as controls. Rat MSC were seeded on these surfaces and cultured for up to 20 d in osteogenic media. Surface topography was characterized by atomic force microscopy, X-ray photoelectron spectroscopy, and static contact angle. Cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium content were measured to evaluate the ability of MSC to adhere and differentiate on PPy-coated TCP. Increased monomer concentrations resulted in PPy films of increased thickness and surface roughness. PPy films generated by different monomer concentrations induced drastically different cellular events. A wide spectrum of cell attachment characteristics (from excellent cell attachment to the complete inability to adhere) were obtained by varying the monomer concentration from 20 m to 50 x 10(-3) M. In particular the 20 x 10(-3) M PPy thin films demonstrated superior induction of MSC osteogenicity, which was comparable to standard TCP dishes, unlike PPy films of similar thickness prepared by chemical polymerization without surfactant. Adhesion of mesenchymal stem cells on tissue culture plates (TCP) coated with polypyrrole thin films made by admicellar polymerization.

In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.

The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups. A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats. Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response. Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups. Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups). Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%). They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation. There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group. However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site. They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 944-951, 2003)

This study describes a method for the quantification of active ligand surface concentration for bulk-modified hydrogels. Two poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) block copolymers were synthesized with terminal poly(ethylene glycol) (PEG) chains of number average molecular weight 1960 and 5190 g/mol. Hydrogels were synthesized with bulk-modified biotin as a model ligand, making use of a PEG spacer arm with a molecular weight of 3400 g/mol. Bulk concentration of biotin was calculated from the initial concentration of biotin, sol fraction, equilibrium water content, and relative incorporation of the polymers to the hydrogel. Surface concentration of biotin bulk-modified hydrogels was quantified with an enzyme linked immunosorbent assay using mouse monoclonal anti-biotin antibody (IgG), horseradish peroxidase-conjugated anti-mouse IgG, and a chemiluminescent substrate. The larger size of the IgG relative to the mesh size of the hydrogels allowed for the quantification of the active biotin at the surface of the hydrogels. Luminescent imaging was used to qualitatively show the isolation of the horseradish peroxidase-conjugated antibodies to the surface of the bulk-modified hydrogel. The active biotin ligands at the surface of hydrogels synthesized with terminal PEG chains of 1960 g/mol were at the top 7.2 nm while for those synthesized with terminal PEG chains of 5190 g/mol were at the top 4.4 nm of the bulk-modified hydrogel. The relationship between bulk ligand concentration and the active ligand concentration at the surface was dependent on the hydrogel composition. The relative magnitude of the PEG spacer arm of the ligand compared to the PEG block length of the copolymer affected the surface availability of the ligand. The results suggest that steric hindrances caused by mobile PEG chains of the copolymer of molecular weight greater than that of the PEG spacer arm contributed to the decreased surface concentration of ligand. This work relates the bulk concentration of a ligand to its surface concentration, an important parameter for the adhesion, migration, and function of anchorage dependent cells.

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 87-95, 2003)

The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.

Several different bioreactors have been investigated for tissue-engineering applications. Among these bioreactors are the spinner flask and the rotating wall vessel reactor. In addition, a new type of culture system has been developed and investigated, the flow perfusion culture bioreactor. Flow perfusion culture offers several advantages, notably the ability to mitigate both external and internal diffusional limitations as well as to apply mechanical stress to the cultured cells. For such investigation, a flow perfusion culture system was designed and built. This design is the outgrowth of important design requirements and incorporates features crucial to successful experimentation with such a system.

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh., (Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 235-241, 2003)

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds., (Copyright 2002 Wiley Periodicals, Inc.)

Bone is an extremely complex tissue that provides many essential functions in the body. Bone tissue engineering holds great promise in providing strategies that will result in complete regeneration of bone and restoration of its function. Currently, such strategies include the transplantation of highly porous scaffolds seeded with cells. Prior to transplantation the seeded cells are cultured in vitro in order for the cells to proliferate, differentiate and generate extracellular matrix. Factors that can affect cellular function include the cell-biomaterial interaction, as well as the biochemical and the mechanical environment. To optimize culture conditions, good understanding of these parameters is necessary. The new developments in bone biology, bone cell mechanotransduction, and cell-surface interactions are reviewed here to demonstrate that bone mechanotransduction is strongly influenced by the biomaterial properties.