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The importance of new processing techniques in tissue engineering
The use of polymer scaffolds in tissue engineering is reviewed and processing techniques are examined. The discussion of polymer-scaffold processing explains fiber bonding, solvent casting and particulate leaching, membrane lamination, melt molding, polymer/ceramic fiber composite-foam processing, phase separation, and high-pressure processing.
Biomaterials and bone mechanotransduction
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.
TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function
BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production. CONCLUSIONS: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro. CLINICAL RELEVANCE: Controlled release of TGF-beta1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.
Cell movement is guided by the rigidity of the substrate
Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.
Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers
Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.
Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery
Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.
Evaluation of transit-time and electromagnetic flow measurement in a chronically instrumented nonhuman primate model
The Physiology Research Branch at Brooks AFB conducts both human and nonhuman primate experiments to determine the effects of microgravity and hypergravity on the cardiovascular system and to identify the particular mechanisms that invoke these responses. Primary investigative efforts in our nonhuman primate model require the determination of total peripheral resistance, systemic arterial compliance, and pressure-volume loop characteristics. These calculations require beat-to-beat measurement of aortic flow. This study evaluated accuracy, linearity, biocompatability, and anatomical features of commercially available electromagnetic (EMF) and transit-time flow measurement techniques. Five rhesus monkeys were instrumented with either EMF (3 subjects) or transit-time (2 subjects) flow sensors encircling the proximal ascending aorta. Cardiac outputs computed from these transducers taken over ranges of 0.5 to 2.0 L/min were compared to values obtained using thermodilution. In vivo experiments demonstrated that the EMF probe produced an average error of 15% (r = .896) and 8.6% average linearity per reading, and the transit-time flow probe produced an average error of 6% (r = .955) and 5.3% average linearity per reading. Postoperative performance and biocompatability of the probes were maintained throughout the study. The transit-time sensors provided the advantages of greater accuracy, smaller size, and lighter weight than the EMF probes. In conclusion, the characteristic features and performance of the transit-time sensors were superior to those of the EMF sensors in this study.
Carbon offers advantages as implant material in human body
Because of such characteristics as high strength and long-term biocompatability, aerospace carbonaceous materials may be used as surgical implants to correct pathological conditions in the body resulting from disease or injury. Examples of possible medical uses include bone replacement, implantation splints and circulatory bypass implants.
Ion beam sputter modification of the surface morphology of biological implants
The surface chemistry and texture of materials used for biological implants may significantly influence their performance and biocompatibility. Recent interest in the microscopic control of implant surface texture has led to the evaluation of ion beam sputtering as a potentially useful surface roughening technique. Ion sources, similar to electron bombardment ion thrusters designed for propulsive applications, are used to roughen the surfaces of various biocompatible alloys or polymer materials. These materials are typically used for dental implants, orthopedic prostheses, vascular prostheses, and artificial heart components. Masking techniques and resulting surface textures are described along with progress concerning evaluation of the biological response to the ion beam sputtered surfaces.
Ion-beam-sputter modification of the surface morphology of biological implants
The surface chemistry and texture of materials used for biological implants may significantly influence their performance and biocompatibility. Recent interest in the microscopic control of implant surface texture has led to the evaluation of ion-beam sputtering as a potentially useful surface roughening technique. Ion sources, similar to electron-bombardment ion thrusters designed for propulsive applications, are used to roughen the surfaces of various biocompatible alloys or polymer materials. These materials are typically used for dental implants, orthopedic prostheses, vascular prostheses, and artificial heart components. Masking techniques and resulting surface textures are described along with progress concerning evaluation of the biological response to the ion-beam-sputtered surfaces.
DF-1, A Nontoxic Carbon Fullerene Based Antioxidant, is Effective as a Biomedical Countermeasure Against Radiation
A long-term goal of radiation research is the mitigation of inherent risks of radiation exposure. Thus the study and development of safe agents, whether biomedical or dietary, that act as effective radioprotectors is an important step in accomplishing this long-term goal. Some of the most effective agents to date have been aminothiols and their derivatives. Unfortunately, most of these agents have side effects such as nausea, vomiting, hypotension, weakness, and fatigability. For example, nausea and emesis occur in most patients treated with WR-2721 (Amifostine), requiring the use of effective antiemetics, with hypotension being the dose-limiting side effect in patients treated. Clearly, the need for a radioprotector that is both effective and safe still exists. Development of biocompatible nano-materials for radioprotection is a promising emerging technology that could be exploited to address the need to minimize biological effects when exposure is unavoidable. Testing free radical scavenging nanoparticles for potential use in radioprotection is exciting and highly relevant. Initial investigations presented here demonstrate the ability of a particular functionalized carbon fullerene nanoparticle, (DF-1), to act as an effective radioprotector. DF-1 was first identified as the most promising candidate in a screen of several functionalized carbon fullerenes based on lack of toxicity and antioxidant therapeutic potential against oxidative injuries (i.e. organ reperfusion and ionizing radiation). Subsequently, DF-1 has been shown to reduce chromosome aberration yield and cell death, as well as overall ROS levels in human lymphocytes and fibroblasts after exposure to gamma radiation and energetic protons while demonstrating no associated toxicity. The dose-reducing factor of DF-1 at LD50 is nearly 2.0 for gamma radiation. In addition, DF-1 treatment also significantly prevented cell cycle arrest after exposure. Finally, DF-1 markedly attenuated COX2 upregulation in cell culture after irradiation thus preventing an inflammatory response to irradiation. Taken together, these results suggest that DF-1 provides potent protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, inflammation and cell death.
Repairing Fractured Bones by Use of Bioabsorbable Composites
A proposed method of surgical repair of fractured bones would incorporate recent and future advances in the art of composite materials. The composite materials used in this method would be biocompatible and at least partly bioabsorbable: that is, during the healing process following surgery, they would be wholly or at least partly absorbed into the bones and other tissues in which they were implanted. Relative to the traditional method, the proposed method would involve less surgery, pose less of a risk of infection, provide for better transfer of loads across fracture sites, and thereby promote better healing while reducing the need for immobilization by casts and other external devices. One requirement that both the traditional and proposed methods must satisfy is to fix the multiple segments of a broken bone in the correct relative positions. Mechanical fixing techniques used in the traditional method include the use of plates spanning the fracture site and secured to the bone by screws, serving of wire along the bone across the fracture site, insertion of metallic intramedullary rods through the hollow portion of the fractured bone, and/or inserting transverse rods through the bone, muscle, and skin to stabilize the fractured members. After the bone heals, a second surgical operation is needed to remove the mechanical fixture(s). In the proposed method, there would be no need for a second surgical operation. The proposed method is based partly on the observation that in the fabrication of a structural member, it is generally more efficient and reliable to use multiple small fasteners to transfer load across a joint than to use a single or smaller number of larger fasteners, provided that the stress fields of neighboring small fasteners do not overlap or interact. Also, multiple smaller fasteners are more reliable than are larger and fewer fasteners. However, there is a trade-off between structural efficiency and the cost of insertion time and materials. The proposed method is further based partly on the conjecture that through-the-thickness reinforcements could be excellent for fixing bone segments for surgical repair. The through-the-thickness reinforcements would superficially resemble nails in both form and function. Denoted small-diameter rods (SDRs) to distinguish them from other narrow rods, these reinforcements would be shot or otherwise inserted through adjacent segments of fractured bone to fix them in their correct relative positions (see figure). Shot insertion would be effected by use an applicator that would amount to a miniaturized and highly refined version of the pneumatic guns often used in carpentry to drive nails and brads. The applicator, envisioned to be about the size of a ball-point-pen, would be driven by pressurized carbon dioxide. To further promote stabilization of the segments, layers of bone glue could be applied to the fracture surfaces prior to insertion of the SDRs. The bone glue could be therapeutically loaded with chemicals to promote growth of bone and fight infection
RF Characterization of a Photocurable PEDOT:PSS:PEGDA Conductive Biomaterial for 3D-Printing Implantable Antennas
In this work, we demonstrate photocurable PEDOT:PSS:PEGDA biomaterial as a promising candidate for intracorporeal 3D printing. Intracorporeal 3D printing offers a less invasive method which offer flexibility to tailor the form factor of printed structures. Previous work has demonstrated the feasibility of a robotic probe to 3D-print biological tissues intracorporeally via a minor incision. This same probe could also 3D-print implantable antennas as long as a suitable conductive material is identified in terms of conductivity, biocompatibility, and ability to cure at room/body temperature for safety purposes. We assess the frequency-dependent conductivity of this biomaterial and explore the Radio-Frequency (RF) performance of resulting antennas operating in free-space and inside tissue-emulating phantoms. Results show that PEDOT:PSS biomaterial with 21% and 30% PEGDA content exhibit a conductivity of ~10 4 S/m up to 5 GHz, suitable for wireless implants. Comparing the two, 21% PEGDA content exhibits poorer curing abilities, while 30% PEGDA exhibits slightly lower conductivity. Measurements for 2.4 GHz free-space dipoles conducted in an anechoic chamber reveal only ~0.8 dB and ~1 dB lower gain for PEDOT:PSS:21%PEGDA and PEDOT:PSS:30%PEGDA biomaterial, respectively, as compared to their copper counterpart. For a 5 mm-deep implanted patch antenna, these two biomaterials exhibit 3.05 dB and 3.84 dB higher transmission loss than copper, respectively. If deemed necessary, this performance degradation can be overcome by increasing the overall antenna size since the printing process is now minimally invasive and miniaturization requirements can be relaxed.
Rodent Biocompatibility Test Using the NASA Foodbar and Epoxy EP21LV
Epoxy has been used successfully to affix NASA foodbars to the inner walls of the Animal Enclosure Module for past space flight experiments utilizing rodents. The epoxy used on past missions was discontinued, making it necessary to identify a new epoxy for use on the STS-108 and STS-107 missions. This experiment was designed to test the basic biocompatibility of epoxy EP21LV with male rats (Sprague Dawley) and mice (Swiss Webster) when applied to NASA foodbars. For each species, the test was conducted with a control group fed untreated foodbars and an experimental group fed foodbars applied with EP21LV. For each species, there were no group differences in animal health and no statistical differences (P<0.05) in body weights throughout the study. In mice, there was a 16% increase in heart weight in the epoxy group; this result was not found in rats. For both species, there were no statistical differences found in other organ weights measured. In rats, blood glucose levels were 15% higher and both total protein and globulin were 10% lower in the epoxy group. Statistical differences in these parameters were not found in mice. For both species, no statistical differences were found in other blood parameters tested. Food consumption was not different in rats but water consumption was significantly decreased 10 to 15% in the epoxy group. The difference in water consumption is likely due to an increased water content of the epoxy-treated foodbars. Finally, both species avoided consumption of the epoxy material. Based on the global analysis of the results, the few parameters found to be statistically different do not appear to be a physiologically relevant effect of the epoxy material, We conclude that the EP21LV epoxy is biocompatible with rodents.
Total Dose Effects (TDE) of heavy ionizing radiation in fungus spores and plant seeds: Preliminary investigations
The opportunity to compare cosmic radiation effects caused during long and short duration exposure flights in biological objects are limited until now, and data obtained so far are very rare and insufficient. Because of the very long exposure of the experiment during the Long Duration Exposure Facility (LDEF) mission (approximately 2000 days) structural changes of the hardware material can be expected which will influence its biocompatibility and, thus, will interact with the radiobiological effects. The aim of the experiment flown on LDEF was a detailed investigation of biological effects caused by cosmic radiation especially of particles of high atomic number Z and high energy. The flight hardware consisted of standard BIOSTACK containers; in these containers a special sandwich construction consisted of visual plastic detectors with seed rsp. spore layers interlocked.
Electrochemically Controlled Reconstitution of Immobilized Ferritins for Bioelectronic Applications
Site-specific reconstituted nanoparticles were fabricated via electrochemically-controlled biomineralization through the immobilization of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, and the electrocatalytic reduction of oxygen on the reconstituted Pt-cored ferritins. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of site-specific electrochemical biomineralization with a protein cage loads ferritins with different core materials. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. This first demonstration of electrochemically controlled site-specific reconstitution of biomolecules provides a new tool for biomineralization and opens the way to produce the bio-templated nanoparticles by electrochemical control. The nanosized platinum-cored ferritins on gold displayed good catalytic activity for the electrochemical reduction of oxygen, which is applicable to biofuel cell applications. This results in a smaller catalyst loading on the electrodes for fuel cells or other bioelectronic devices.
Physical and Biological Properties of Cobalt- and Copper-Doped Calcium Phosphates as Bone Substitute Materials
Arthritis, osteoporosis, and other bone diseases and defects are common medical issues worldwide. By utilizing ceramic biomaterials as bone substitutes to treat these diseases, bone regeneration can be promoted and toxicity from the substitute material can be limited. Calcium phosphate (CaP) ceramics have become popular as bone substitutes due to their biocompatibility and similarity in composition to natural bone. The purpose of this research was to investigate the physical properties and biological responses of CaP-based bone substitutes, doped with metal ions. Some metal ions are present in natural bone in small amounts, and recent studies show they affect the biological responses of CaPs significantly. Dopants such as magnesium (Mg), strontium (Sr), silicon (Si) and iron (Fe) alter the phase composition, mechanical properties, osteogenesis and angiogenesis properties of CaPs, depending on their concentration and method of addition. In the present study, the effects of cobalt (Co) and copper (Cu) on the physical and biological properties of two main CaP materials, brushite cement (BrC) and tricalcium phosphate (TCP), have been investigated. Different concentrations of dopants in forms of oxide and/or chloride were selected and their effect on phase composition, sintering behavior, density, setting time, compressive strength and in vitro interaction with osteosarcoma and osteoblast cells were studied. Different techniques of sample preparation, challenges during cement preparation and compact sample pressing, sintering, and methods of dopant addition are discussed. The presence of Cu in tricalcium phosphate was found to increase thermal stability of the material and decrease the impact of sintering temperature on porosity. In addition, Cu caused a reduction in expression of inflammatory gene markers by human osteoblast cells and an increased expression at early time points due of osteoinductive markers.The addition of Cu and Co to brushite cement caused an increase in thermal stability of the material, and the addition of Cu caused significant increase in setting time. Small dopant amounts of Co caused decrease in setting time, but higher amounts caused an increase. In addition, Cu dopant in small amounts resulted in an increase of compressive strength. Both Cu and Co led to a decrease in expression of inflammatory gene markers by osteoblast and osteosarcoma cells respectively, and Cu also caused an increase in osteoinductive marker expression. Thus, these results indicate that Cu and Co have a positive impact on the physical, mechanical and biological properties of calcium phosphate biomaterials.