Biomedical subjects

D A Puleo

Publications and source records attributed to D A Puleo.

At least 19 recordsLinked to original sources

PubMed2006

Modulated release of bioactive protein from multilayered blended PLGA coatings.

The objective of this study was to develop a poly(D,L-lactic-co-glycolic acid) (PLGA)-based coating system for producing biologically-inspired delivery profiles. Protein-loaded microspheres were made from PLGA (50:50) terminated with carboxylic acid groups (PLGA-2A) blended either with more hydrophobic PLGA (50:50) having lauryl ester endcaps (PLGA-LE) or with the more hydrophilic Pluronic F-127 (PF-127). Dense coatings were formed by pressure-sintering the microspheres. Altering hydrophobicity changed the water concentration within coatings, and consequently the time to onset of polymer degradation and protein release was modulated. After blending up to 8% Pluronic, degradation by-products began accumulating immediately upon incubation in saline, whereas, degradation was delayed for up to 14 days with blending of up to 30% PLGA-LE. Primary protein release peaks from one-layer coatings could be created from 7 to 20 days using 8% PF-127 or 30% PLGA-LE blends, respectively. Multilayered coatings of different blends generated several release peaks, with their temporal occurrence remaining approximately the same when layers of other hydrophobicity were added above or below. To allow design of coatings for future use, results were used to construct a model based on Fourier analysis. This polymer blend system and model can be used to mimic temporally varying profiles of protein expression.

Chemistry, Pharmaceutical↗

PubMed2006

Mechanical and degradation behavior of polymer-calcium sulfate composites.

Calcium sulfate (CS) is one of the oldest bone graft materials still in use. Its main limitations are poor handling characteristics, poor mechanical properties, and a resorption rate that is too fast for some applications. The present study investigated the effect of viscous polymers, such as carboxymethylcellulose (CMC) and hyaluronan (HY), on the handling characteristics, mechanical properties, and degradation behavior of CS. CMC and HY were added to CS at concentrations from 1-10 wt%. Addition of CMC to CS at more than 4 wt% produced a putty-like material and decreased the density of the composite, while also increasing flexural and compressive strength at higher loadings. Incorporation of CMC produced a concentration-dependent increase in water absorption and degradation rate. At an equivalent loading, HY-containing CS composites showed better compressive strength than CS with CMC. Overall, addition of CMC or HY to CS resulted in composite materials with better handling characteristics and improved mechanical properties after set, however the degradation rate of the augmented materials was increased. These properties suggest that the enhanced CS materials may be useful in certain clinical situations, such as filling non-uniform bone defects and situations that require mechanical integrity of the bone graft substitute during implantation.

Biocompatible Materials↗

PubMed2004

Cell responses to BMP-2 and IGF-I released with different time-dependent profiles.

During wound healing, growth factors are expressed in time-dependent amounts. Constant delivery of biomolecules, however, is often used to influence cell and tissue behavior. In the present studies, a crosslinked gelatin-coating system was used to deliver bone morphogenetic protein 2 (BMP-2) or insulin-like growth factor (IGF-I) to three types of mesenchymal cells with three temporally varying release profiles. The "early" delivery profile released most of the growth factor within the first 2 days. The "pseudo-zero-order" profile approximated constant rate of delivery for about 5 days. The "late" delivery profile released most of the growth factor after about 5 days. Early delivery of IGF-I had the greatest effect on mitogenesis of SaOS-2 human osteosarcoma cells with a secondary effect noted nearly 5 days after delivery was completed. Late delivery of BMP-2 resulted in greatest alkaline phosphatase (AP) activity in mouse pluripotent C3H10T1/2 cells. Rat bone marrow stromal cells (BMCs) responded to all delivery profiles of BMP-2, with the duration of elevated AP activity increasing as the amount of BMP-2 delivered increased. In addition to an early increase in AP activity, late release also stimulated BMCs over a longer portion of the culture period. BMCs responded similarly to SaOS-2 cells when seeded on early IGF-I delivery coatings, increasing AP activity after delivery had ended. Overall, these studies further show the importance of delivery profile, specifically the characteristics of time and concentration, on cell and tissue responses.

Animals↗

PubMed2004

In vitro effects of combined and sequential delivery of two bone growth factors.

Bone formation and repair occur by a complex cascade involving numerous growth factors and cytokines. In this study, two-layered heterogeneously loaded and crosslinked gelatin coatings were used to obtain combined and sequential delivery of two bone growth factors, BMP-2 and IGF-I, in cell cultures. Peak release from the top and bottom layers was localized around 1 and 6 days, respectively. For comparison, cells were also treated with soluble growth factors directly added to the culture medium. Pluripotent C3H10T1/2 (C3H) cells responded to soluble growth factor treatments with the greatest specific alkaline phosphatase (AP) activity resulting from addition of BMP-2 followed by IGF-I or by BMP-2+IGF-I. Altered loading and subsequent release of BMP-2 and IGF-I from gelatin coatings also affected AP activity in C3H cultures, and the coatings influenced AP activity and incorporation of calcium in the extracellular matrix of bone marrow stromal cell cultures. Early delivery of BMP-2 followed by increased release of BMP-2 and IGF-I after 5 days resulted in the largest, as well as earliest, elevation of AP activity and mineralized matrix formation compared to controls and other treatments. Simultaneous release of both growth factors from both layers did not significantly change AP activity or matrix calcium content compared to control coatings. These results demonstrate that temporally varying delivery of multiple growth factors can significantly affect cell behavior.

Animals↗

PubMed2002

A technique to immobilize bioactive proteins, including bone morphogenetic protein-4 (BMP-4), on titanium alloy.

Immobilization of biomolecules on surfaces enables both localization and retention of molecules at the cell-biomaterial interface. Since metallic biomaterials used for orthopedic and dental implants possess a paucity of reactive functional groups, biomolecular modification of these materials is challenging. In the present work, we investigated the use of a plasma surface modification strategy to enable immobilization of bioactive molecules on a "bioinert" metal. Conditions during plasma polymerization of allyl amine on Ti-6Al-4V were varied to yield 5 ("low")- and 12 ("high")-NH2/nm2. One- and two-step carbodiimide schemes were used to immobilize lysozyme, a model biomolecule, and bone morphogenetic protein-4 (BMP-4) on the aminated surfaces. Both schemes could be varied to control the amount of protein bound, but the one-step method destroyed the activity of immobilized lysozyme because of crosslinking. BMP-4 was then immobilized using the two-step scheme. Although BMP bound to both low- and high-NH2 surfaces was initially able to induce alkaline phosphatase activity in pluripotent C3H10T1/2 cells, only high amino group surfaces were effective following removal of weakly bound protein by incubation in cell culture medium.

Adsorption↗

PubMed2001

Triphasic release model for multilayered gelatin coatings that can recreate growth factor profiles during wound healing.

Multilayered gelatin coatings were created to mimic growth factor profiles that normally occur during fracture healing. A model was developed to relate crosslinking and loading of individual layers to protein release. Modeling was simplified by dividing release profiles into three phases. The diffusion-controlled phase was determined by calculating periods of constant diffusivity for each homogeneous layer within devices. Diffusivity was a power law function of crosslinking. Fick's second law of diffusion was then used to determine release during the diffusion-controlled phase. Secondary diffusivity was determined by summing resistances of each successive homogeneous layer. The initial burst phase was defined as events proceeding the diffusion-controlled phase. Percentage of drug burst was a linear function of crosslinking. Release during the degradation-controlled phase, events following diffusion-controlled phase, was estimated based on first order hydrolysis of crosslinks. The model predicted time-variant release of differently labeled protein measured experimentally, and it can be used to design coatings to recreate the cascade of biomolecules that determine natural bone repair.

Animals↗

PubMed1999

Understanding and controlling the bone-implant interface.

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone-biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone-implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone-implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue-implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone-implant interface.

Adsorption↗

PubMed1999

Release and retention of biomolecules in collagen deposited on orthopedic biomaterials.

Delivery of osteotropic biomolecules directly to the bone-implant interface can alter initial interactions between tissue and biomaterial. To this end, type I collagen coatings containing a model biomolecule, lysozyme, were deposited on Co-Cr-Mo and Ti-6Al-4V. Two deposition methods were examined. In the first, lysozyme was deposited concurrently with collagen, while in the second, protein was impregnated into previously deposited collagen coatings. The amount of collagen and the amount of lysozyme loaded into collagen were varied to provide different amounts of weakly and strongly bound protein. Release and retention of lysozyme were monitored over a 7 d period of incubation in physiological saline. For both methods, larger amounts of collagen in the coatings allowed incorporation of more lysozyme. Additionally, loading collagen coatings with greater amounts of lysozyme resulted in release of more protein. During the first 24-96 h of incubation, loosely bound protein was eluted, resulting in release of 2 micrograms to 55 mg (5-75% of the amount available) of enzymatically active lysozyme. This left 25-95% of the protein bound to the collagen-coated biomaterials and, thus, available for later release during degradation of the collagen.

Animals↗

PubMed1998

In vitro cellular responses to bioerodible particles loaded with recombinant human bone morphogenetic protein-2.

Porous 50:50 poly(d,l lactide-co-glycolide) microspheres containing varying amounts of "free" recombinant human bone morphogenetic protein-2 (rhBMP-2) were evaluated for their ability to induce/enhance expression of osteoblastic characteristics by pluripotent mesenchymal cells in vitro. "Free" protein (Fp) is defined as protein present on the surface and within the porous matrix of the microspheres. Four preparations of bioerodible particles (BEP) were used: blank--without rhBMP-2; low Fp--24 microg of free rhBMP-2 per g of particles; medium Fp--403 microg/g; and high Fp--884 microg/g. C3H10T1/2 cells (C3H) and bone marrow stromal cells (BMC) were cultured with 1 mg of BEP for up to 4 weeks, and cell growth and expression of osteogenic responses were determined weekly. For both cell types, control cultures (neither BEP nor rhBMP-2) and cultures with blank BEP exhibited no or minimal osteoblastic characteristics. Compared to control and blank BEP cultures, C3H cells responded to particles having medium and high amounts of free rhBMP-2 with increased cell growth and alkaline phosphatase activity, but osteocalcin secretion and mineralization were not markedly influenced. Low Fp BEP enhanced only the alkaline phosphatase activity of C3H cells. In contrast, although growth was not affected, rhBMP-2-loaded BEP increased alkaline phosphatase activity, osteocalcin secretion, and mineralization in BMC cultures in a dose-dependent manner (i.e., blank < low < medium < high Fp).

Animals↗

PubMed1997

Effect of metal ions on the formation and function of osteoclastic cells in vitro.

To determine if metal ions play a contributing role in loosening of orthopedic implants, the present work investigated whether sublethal concentrations of ions affect the formation and function of osteoclasts in vitro. Rat bone marrow cells were cultured on slices of devitalized bone and in the presence of ions associated with Co-Cr-Mo and Ti-6A1-4V alloys for up to four weeks. Cultures were assayed for total intracellular protein, used as measure of cell growth, and resorption activity of osteoclastic cells derived from hematopoietic stem cells was quantified using image analysis. Although Co2+ caused delayed toxicity not previously observed during short-term experiments, none of the other ions affected cell proliferation, indicating that the chosen concentrations were sublethal. In general, exposure of bone marrow cultures to ions caused either a decrease or no change in the total area of bone resorption. A decrease in the number of resorption pits formed by osteoclastic cells was primarily responsible for the decrease in total amount of resorption. Therefore, even though cells continued to grow over the entire culture period, less osteoclastic activity was observed. Findings indicate that if metal ions play a role in periprosthetic pathology, they may contribute to implant failure by impairing bone repair while allowing fibrous tissue formation following debris-induced osteolysis.

Alloys↗

PubMed1997

Retention of enzymatic activity immobilized on silanized Co-Cr-Mo and Ti-6Al-4V.

Biochemical surface modification of biomaterials utilizes immobilized biomolecules to induce preferred tissue responses. Operational stability, or retention of biological activity, of biochemically modified biomaterials is a fundamental determinant of their usefulness. The present study investigated retention of enzymatic activity immobilized on silanized Co-Cr-Mo and Ti-6Al-4V. A model enzyme, trypsin, was immobilized on monolayers and multilayers of silane deposited from aqueous or organic solutions of gamma-aminopropyltriethoxysilane (APS). Trypsin-conjugated biomaterials were incubated in cell culture medium at 37 degrees C for up to 96 h, and the residual immobilized activity was measured. Retention of bioactivity in physiological saline was dependent on the type of material and on the method of silanization. Activity of enzyme adsorbed on the metals was lost within 24-48 h. Both mono- and multilayers of APS deposited on Co-Cr-Mo by aqueous silanization effectively retained enzymatic activity throughout the 96 h incubation period. The monolayer retained approximately 23% of the activity initially present, and the multilayers retained approximately 50% of the initial activity. Organic silanization of Co-Cr-Mo was marginally effective as it initially slowed the loss of activity. However, all activity was lost by 48-72 h of incubation. Neither organic nor aqueous silanization enhanced retention of enzymatic activity on Ti-6Al-4V.

Alloys↗

PubMed1997

Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro.

Bone morphogenetic proteins (BMPs) induce osteoblastic responses in cultures of pluripotent mesenchymal cells. The effects of chronic treatment of these cells with BMPs and of withdrawal following exposure, however, have not been fully elucidated. Thus, the aim of this study was to obtain information about the duration of exposure to recombinant human BMP-2 (rhBMP-2) required for expression and retention of osteoblastic characteristics with subsequent formation of a mineralized extracellular matrix in mesenchymal cell cultures. C3H1OT1/2 cells and bone marrow stromal cells were cultured with 1 mug/ml rhBMP-2 for either 0, 7, 14, 21, or 28 days, with the remainder of the 4 week total culture period in the absence of rhBMP-2. Growth and expression of osteoblastic characteristics were examined at the end of each week. C3H1OT1/2 cells responded to increasing duration of exposure to rhBMP-2 with increased cell growth. Additionally, the longer the cells were exposed to rhBMP-2, the more fully they expressed and sustained osteoblastic traits, i.e., they exhibited duration of exposure-dependent higher levels of alkaline phosphatase and osteocalcin and larger total amounts of mineral in the matrix. In comparison, exposure of bone marrow stromal cells to rhBMP-2 for at least 14 days restrained cell growth and prevented detachment. With respect to osteoblastic traits, stromal cells exposed to rhBMP-2 also exhibited a dependence on the duration of exposure, however, cultures treated for 14, 21, or 28 days exhibited similar levels of alkaline phosphatase activity and comparable amounts of calcium in the mineralizing matrix.

Alkaline Phosphatase↗

PubMed1997

Biochemical surface modification of Ti-6Al-4V for the delivery of protein to the cell-biomaterial interface.

Biochemical surface modification involves delivery of biomolecules to the tissue-implant interface to induce desired cell and tissue responses. We have previously had success in immobilizing and retaining bioactive molecules on Co-Cr-Mo but not on Ti-6Al-4V. The purpose of this study was to modify the gamma-aminopropyltriethoxysilane (APS) scheme to enable successful attachment of protein to the surface of Ti-6Al-4V. Ti-6Al-4V samples were silanized with organic (acetone) solutions of APS and dried at increasing temperatures. Concentrations resulting in 2-4 NH2 per nm2 of nominal surface area were incubated in physiological saline for up to 96 hr to assess retention of amino groups. A model protein, trypsin, was coupled to silanized Ti-6Al-4V via glutaraldehyde. The samples were then incubated in saline, and the activity of residual immobilized enzyme was quantified. After drying at 45, 80, or 115 degrees C, the NH2 groups were lost from the surface by 24 hr of incubation in saline. On samples dried at 150 degrees C, with 4% APS, the number of NH2 groups increased after 8 hr and remained relatively constant through 96 hr. Likewise, at 150 degrees C with 2% APS the surface density of NH2 groups reached a maximum at 24 hr and remained relatively constant up to 96 hr. When incubated for 96 hr, Ti-6Al-4V with 4% APS and dried at 150 degrees C retained approximately 31% of the activity initially immobilized, whereas protein on 45 degrees C or adsorbed samples was lost by 24-48 hr.

Alloys↗

PubMed1996

Use of p-nitrophenyl chloroformate chemistry to immobilize protein on orthopedic biomaterials.

Biochemical surface modification involves covalently immobilizing biomolecules onto biomaterial surfaces to induce specific biological responses. This approach may be useful for enhancing the fixation of orthopedic implants. p-Nitrophenyl chloroformate (p-NPC) was used to immobilize protein on bulk samples of Co-Cr-Mo and Ti-6Al-4V. Activation of both materials was dependent on the concentration of p-NPC, with a maximum of approximately 1.5 active groups/nm2 of nominal surface area. Trypsin was used as a model protein because much is known about its structure and mode of action. Derivatization with 0.65 mg p-NPC/cm2 resulted in significantly greater enzymatic activity (7.4 BAEE [N-(alpha)-benzoyl-L-arginine ethyl ester hydrochloride] units) on the Co-Cr-Mo samples compared with higher concentrations of p-NPC (5 BAEE units) and with simple adsorption of trypsin (1.5 BAEE units). An activity of 10.5 BAEE units was measured on both adsorbed and p-NPC-activated Ti-6Al-4V, with the exception of samples derivatized with 1.95 mg p-NPC/cm2, on which activity was significantly lower (4 BAEE units). In probing the linkages between trypsin and biomaterial by treatment with chaotropic agents, guanidine hydrochloride (GuHCl) was observed to eliminate more enzymatic activity than was urea. On Co-Cr-Mo samples, GuHCl removed nearly all the trypsin activity, while urea significantly decreased the activity only at a concentration of 0.65 mg p-NPC/cm2. Treatment of Ti-6Al-4V samples with GuHCl caused a trend of decreasing activity with increasing concentration of p-NPC, whereas urea had no effect on immobilized trypsin activity.

Adsorption↗

PubMed1996

Ti-6Al-4V ion solution inhibition of osteogenic cell phenotype as a function of differentiation timecourse in vitro.

Metal ions released from the implant surface are suspected of playing some contributing role in loosening of hip and knee prostheses. previous work in this laboratory demonstrated that sublethal doses of the ionic constituents of Ti-6Al-4V alloy suppressed expression of the osteoblastic phenotype and deposition of a mineralized matrix. The purpose of this work was to further explore this suppression as a function of the normal time-course of phenotype expression. Bone marrow stromal cells were harvested from juvenile rats and exposed to time-staggered doses of a solution of ions representing Ti-6Al-4V alloy. Cells were cultured for four weeks and assayed for total protein, alkaline phosphatase, intra-and extracellular osteocalcin, and calcium. Ti-6Al-4V solutions were found to produce little difference from control solutions for total protein or alkaline phosphatase levels, but strongly inhibited osteocalcin synthesis. Calcium levels were reduced when ions were added before a critical point of osteoblastic differentiation (between 2 and 3 weeks after seeding). These results indicate that ions associated with Ti-6Al-4V alloy inhibited the normal differentiation of bone marrow stromal cells to mature osteoblasts in vitro, suggesting that ions released from implants in vivo may contribute to implant failure by impairing normal bone deposition.

Alkaline Phosphatase↗

PubMed1996

Biochemical surface modification of Co-Cr-Mo.

Because of the limited mechanical properties of tissue substitutes formed by culturing cells on polymeric scaffolds, other approaches to tissue engineering must be explored for applications that require complete and immediate ability to bear weight, e.g. total joint replacements. Biochemical surface modification offers a way to partially regulate events at the bone-implant interface to obtain preferred tissue responses. Tresyl chloride, gamma-aminopropyltriethoxysilane (APS) and p-nitrophenyl chloroformate (p-NPC) immobilization schemes were used to couple a model enzyme, trypsin, on bulk samples of Co-Cr-Mo. For comparison, samples were simply adsorbed with protein. The three derivatization schemes resulted in different patterns and levels of activity. Tresyl chloride was not effective in immobilizing active enzyme on Co-Cr-Mo. Aqueous silanization with 12.5% APS resulted in optimal immobilized activity. Activity on samples derivatized with 0.65 mg p-NPC cm-2 was four to five times greater than that on samples simple adsorbed with enzyme or optimally derivatized with APS and was about eight times that on tresylated samples. This work demonstrates that, although different methods have different effectiveness, chemical derivatization can be used to alter the amount and/or stability of biomolecules immobilized on the surface of Co-Cr-Mo.

Adsorption↗

PubMed1996

Stability of trypsin immobilized on inorganic orthopedic biomaterials.

Biochemical surface modification of biomaterials utilizes immobilized biomolecules to induce preferred tissue responses. Although several techniques are available for immobilizing biomolecules on organic substrates, comparatively few are available for use with inorganic materials, such as those used in many orthopedic applications. The present study investigated the stability/elutability of a model enzyme immobilized on Co-Cr-Mo and Ti-6Al-4V alloys using p-nitrophenyl chloroformate (p-NPC). Trypsin-conjugated biomaterials were incubated in cell culture medium at 37 degrees C for up to 96 hr, and the residual immobilized activity was measured. Although all samples initially bound enzymatically active trypsin, significant decreases were observed within the first 2 hr of incubation. Immobilization of trypsin on Co-Cr-Mo treated with 0.65 mg p-NPC/cm2 of nominal surface area gave significantly higher residual activity than on untreated samples at 24-96 hr of incubation and prevented the nearly complete loss of enzymatic activity that was observed with free (not immobilized) enzyme. Derivatization of Ti-6Al-4V with p-NPC was not beneficial to the level of immobilized enzymatic activity after incubation in medium for longer than 6 hr.

Alloys↗

PubMed1995

Acute toxicity of metal ions in cultures of osteogenic cells derived from bone marrow stromal cells.

The effects of metal ions released from orthopedic implants on nearby bone cells remain largely unknown. The purpose of this study was to examine the acute toxicity of metal ions on osteogenic cells derived from bone marrow. Bone marrow stromal cells were cultured with metal ions found in commonly used orthopedic implants, that is, Ti-6Al-4V, Co-Cr-Mo, and 316L stainless steel. Solutions of individual ions and combinations representing the alloy composition were prepared from atomic absorption standards and added to the cultures to give concentrations ranging from 50 ppb to 50 ppm. After a 48-h period of exposure to ions, the bone marrow cultures were examined for effects of cytotoxicity by measuring total cell number, total cell protein, and mitochondrial activity. Cr6+ was grossly cytotoxic; Co2+, Mo6+, Fe3+, and Ni2+ were moderately cytotoxic; and Ti4+, Al3+, V5+, and Mn2+ were minimally toxic, as determined by the assays used. Ion solutions representing Co-Cr-Mo and 316L stainless steel were moderately toxic; solutions representing Ti-6Al-4V were toxic at only the highest concentrations used. The observed cytotoxicity was time-dependent, with irreversible toxic effects being initiated following as short as a 3- to 6-hour exposure. These results show that metal ions associated with Co-Cr-Mo and 316L stainless steel are toxic to osteogenic cells at concentrations approximating those measured in the fibrous membrane encapsulating orthopedic implants.

Animals↗