The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 2

Delivery Systems for the BMPs | April 01, 2001

TGF-β1 Release from Biodegradable Polymer Microparticles: Its Effects on Marrow Stromal Osteoblast Function

Lichun Lu, PhD; Michael J. Yaszemski, MD, PhD; Antonios G. Mikos, PhD

Investigation performed at the Departments of Bioengineering and Chemical Engineering, Rice University, Houston, Texas
Lichun Lu, PhD
Michael J. Yaszemski, MD, PhD
Departments of Orthopedic Surgery and Bioengineering, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, U.S.A.

Antonios G. Mikos, PhD
Department of Bioengineering, Rice University, 6100 Main Street, MS-142, Houston, TX 77005-1892, U.S.A. E-mail address: mikos@rice.edu

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from NIH (R01-AR44381). None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

J Bone Joint Surg Am, 2001 Apr 01;83(1 suppl 2):S82-S92

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Abstract

Background: Controlled release of transforming growth factor-&betabeta;1 (TGF-&betabeta;1) 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-&betabeta;1 delivery and the effects of released TGF-&betabeta;1 on the proliferation and differentiation of marrow stromal cells in vitro.

Methods: Recombinant human TGF-&betabeta;1 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-&betabeta;1 was determined after 3 days in culture. The effects of TGF-&betabeta;1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period.

Results: TGF-&betabeta;1 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-&betabeta;1 released from the microparticles similar to that of added TGF-&betabeta;1, indicating that the activity of TGF-&betabeta;1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-&betabeta;1 dosage of 1.0 ng/ml after 3 days, the released TGF-&betabeta;1 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-&betabeta;1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro.

Clinical Relevance: Controlled release of TGF-&betabeta;1 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.

Figures in this Article

Introduction

Introduction | Materials and Methods | Results | Discussion | References

Many afflictions require the controlled delivery of therapeutic molecules in order for treatment to be effective. Transforming growth factor-&betabeta;1 (TGF-&betabeta;1) has been studied as a potential induction factor for bone tissue engineering^1-6. TGF-&betabeta;1, a member of the TGF-&betabeta; superfamily, is a disulfide-linked homodimer consisting of two 112 amino acid residue polypeptides and has a molecular weight of approximately 25 kDa. It is a multifunctional protein that regulates many aspects of cellular activity including cell proliferation and differentiation and extracellular matrix metabolism in a time and concentration-dependent fashion². It plays a significant role in regulating bone formation in fracture callus⁷ and increases osteoblast proliferation and differentiation during fracture healing in a rat femur model⁸.

Microparticles fabricated from biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) copolymers have been widely utilized as carriers for bioactive molecules^4,9-11. These microparticles can be implanted at an afflicted site during surgery, injected as a suspension to a wound area, or impregnated into polymer scaffolds and then transplanted¹². PLGA has been shown to be biocompatible and biodegradable and has been approved by the FDA for certain human clinical uses¹³. The degradation times of PLGA can be altered from days to years by varying the polymer molecular weight, the ratio of lactic to glycolic acid in the copolymer, or the structure of the microparticles^14,15.

The release rates of bioactive molecules from PLGA microparticles are affected by many factors including the structure, size, and solubility of the encapsulated molecules as well as the composition, molecular weight, structure, and degradation properties of the polymer¹⁴. The combination of poly(ethylene glycol) (PEG) and PLGA to form blend microparticles allows further attenuation of the release profile of the loaded compound¹⁰. Additionally, the release rate is affected by co-encapsulation of other molecules in the microparticle formulations¹⁶. Furthermore, the protein release profiles are dependent on environmental conditions such as the acidity of the release medium. For instance, the inflammatory response following implantation of devices or the release of acidic PLGA degradation products at late stages of microparticle degradation often results in a decrease in local pH^14,17,18. This decrease in pH can affect the structure, solubility, diffusivity, and activity of the loaded compounds.

We have recently developed a novel injectable, in situ polymerizable, biodegradable orthopaedic material based on poly(propylene fumarate) (PPF) for filling skeletal defects¹⁹. PPF, combined with a vinyl monomer (N-vinyl pyrrolidone) and an initiator (benzoyl peroxide), forms an injectable paste that can be polymerized in situ, filling contained skeletal defects of any shape or size¹⁹. Additionally, incorporation of solid phase components including &betabeta;-tricalcium phosphate (&betabeta;-TCP) and sodium chloride can result in a porous composite material possessing mechanical properties sufficient for the replacement of human trabecular bone²⁰. Furthermore, marrow stromal cells can attach, proliferate, and express differentiated osteoblastic function when cultured on PPF/&betabeta;-TCP substrates in vitro²¹. It is an objective of our laboratory to develop an injectable formulation that may provide a vehicle for delivery of microparticle carriers of growth factors to the defect site and induce bone regeneration.

This study investigates the effects of PEG content and buffer pH on the release kinetics of TGF-&betabeta;1 from biodegradable PLGA/PEG blend microparticles and the degradation of PLGA¹⁵, as well as the effects of TGF-&betabeta;1 released from these microparticles on the proliferation and differentiation of rat marrow stromal cells seeded on PPF substrates²².

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Fig. 1-A:: Figs. 1-A and 1-B Cumulative release kinetics of transforming growth factor-&betabeta;1 (TGF-&betabeta;1) from poly(DL-lactic-co-glycolic acid) PLGA/poly(ethylene glycol) (PEG) microparticles as a function of incubation time: microparticles with varied PEG contents in pH 7.4 phosphate buffered saline (PBS) (Fig. 1-A) and microparticles with 5% PEG in different buffers (Fig. 1-B). Error bars represent means ± SD for n = 4. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 1-B:: Figs. 1-A and 1-B Cumulative release kinetics of transforming growth factor-&betabeta;1 (TGF-&betabeta;1) from poly(DL-lactic-co-glycolic acid) PLGA/poly(ethylene glycol) (PEG) microparticles as a function of incubation time: microparticles with varied PEG contents in pH 7.4 phosphate buffered saline (PBS) (Fig. 1-A) and microparticles with 5% PEG in different buffers (Fig. 1-B). Error bars represent means ± SD for n = 4. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 2:: Cumulative release kinetics of transforming growth factor-&betabeta;1 (TGF-&betabeta;1) from poly(DL-lactic-co-glycolic acid) (PLGA)/ poly(ethylene glycol) (PEG) microparticles in pH 7.4 phosphate buffered saline (PBS) for varied initial TGF-&betabeta;1 loading densities. Error bars represent means ± SD for n = 3. (Reproduced by permission from Peter et al. Effects of transforming growth factor-beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates. J Biomed Mater Res. 2000;50:452-62.)

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Fig. 3-A:: Figs. 3-A and 3-B Decrease of weight average molecular weight (Mw) of poly(DL-lactic-co-glycolic acid) (PLGA) in PLGA/poly(ethylene glycol) (PEG) microparticles as a function of incubation time: microparticles with varied PEG contents in pH 7.4 phosphate buffered saline (PBS) (Fig. 3-A) and microparticles with 5% PEG in different buffers (Fig. 3-B). Error bars represent means ± SD for n = 4. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 3-B:: Figs. 3-A and 3-B Decrease of weight average molecular weight (Mw) of poly(DL-lactic-co-glycolic acid) (PLGA) in PLGA/poly(ethylene glycol) (PEG) microparticles as a function of incubation time: microparticles with varied PEG contents in pH 7.4 phosphate buffered saline (PBS) (Fig. 3-A) and microparticles with 5% PEG in different buffers (Fig. 3-B). Error bars represent means ± SD for n = 4. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 4-A:: Figs. 4-A, 4-B, and 4-C Scanning electron micrographs of poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 4-A), after 28 days of incubation in pH 7.4 phosphate buffered saline (PBS) (Fig. 4-B), and after 28 days of incubation in pH 5 buffer (Fig. 4-C). Scale bars are 10 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 4-B:: Figs. 4-A, 4-B, and 4-C Scanning electron micrographs of poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 4-A), after 28 days of incubation in pH 7.4 phosphate buffered saline (PBS) (Fig. 4-B), and after 28 days of incubation in pH 5 buffer (Fig. 4-C). Scale bars are 10 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 4-C:: Figs. 4-A, 4-B, and 4-C Scanning electron micrographs of poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 4-A), after 28 days of incubation in pH 7.4 phosphate buffered saline (PBS) (Fig. 4-B), and after 28 days of incubation in pH 5 buffer (Fig. 4-C). Scale bars are 10 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 5-A:: Figs. 5-A, 5-B, and 5-C Fluorescence micrographs showing the distribution of fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) within poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 5-A), after 28 days of incubation in pH 7.4 PBS (Fig. 5-B), and after 28 days of incubation in pH 5 buffer (Fig. 5-C). Scale bar is 20 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 5-B:: Figs. 5-A, 5-B, and 5-C Fluorescence micrographs showing the distribution of fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) within poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 5-A), after 28 days of incubation in pH 7.4 PBS (Fig. 5-B), and after 28 days of incubation in pH 5 buffer (Fig. 5-C). Scale bar is 20 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 5-C:: Figs. 5-A, 5-B, and 5-C Fluorescence micrographs showing the distribution of fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) within poly(DL-lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles with 5% PEG: before degradation (Fig. 5-A), after 28 days of incubation in pH 7.4 PBS (Fig. 5-B), and after 28 days of incubation in pH 5 buffer (Fig. 5-C). Scale bar is 20 m. (Reproduced by permission from Lu et al. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J Biomed Mater Res. 2000;50:440-51.)

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Fig. 6-A:: Figs. 6-A and 6-B Alkaline phosphatase (ALP) activity of marrow stromal cells after 3 days of in vitro culture on poly(propylene fumarate) (PPF) and tissue-culture polystyrene (TCPS) substrates in the presence of different transforming growth factor-&betabeta;1 (TGF-&betabeta;1) concentrations prepared by the addition of TGF-&betabeta;1 in media (Fig. 6-A) or the incubation of media with TGF-&betabeta;1-loaded poly(DL-lactic-co-glycolic acid) (PLGA)/ poly(ethylene glycol) (PEG) microparticles for 3 days to obtain conditioned media (Fig. 6-B). The ALP activities were normalized to the values for control TCPS in the absence of TGF-&betabeta;1. Error bars represent means ± SD for n = 3. (Reproduced by permission from Peter et al. Effects of transforming growth factor-beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates. J Biomed Mater Res. 2000;50:452-62.)

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Fig. 6-B:: Figs. 6-A and 6-B Alkaline phosphatase (ALP) activity of marrow stromal cells after 3 days of in vitro culture on poly(propylene fumarate) (PPF) and tissue-culture polystyrene (TCPS) substrates in the presence of different transforming growth factor-&betabeta;1 (TGF-&betabeta;1) concentrations prepared by the addition of TGF-&betabeta;1 in media (Fig. 6-A) or the incubation of media with TGF-&betabeta;1-loaded poly(DL-lactic-co-glycolic acid) (PLGA)/ poly(ethylene glycol) (PEG) microparticles for 3 days to obtain conditioned media (Fig. 6-B). The ALP activities were normalized to the values for control TCPS in the absence of TGF-&betabeta;1. Error bars represent means ± SD for n = 3. (Reproduced by permission from Peter et al. Effects of transforming growth factor-beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates. J Biomed Mater Res. 2000;50:452-62.)

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Figs. 7-A, 7-B, 7-C, and 7-D:: Figs. 7-A, 7-B, 7-C, and 7-D Marrow stromal cells were assayed for ³H-thymidine uptake (Fig. 7-A), total cell number (Fig. 7-B), alkaline phosphatase (ALP) activity (Fig. 7-C), and osteocalcin production (Fig. 7-D) during 21 days of in vitro culture on poly(propylene fumarate) (PPF) substrates in the presence of transforming growth factor-&betabeta;1 (TGF-&betabeta;1)-loaded poly(DL-lactic-co-glycolic acid) (PLGA)/ poly(ethylene glycol) (PEG) microparticles. PPF substrates with blank PLGA/PEG microparticles, PPF, and tissue-culture polystyrene (TCPS) served as controls. Error bars represent means ± SD for n = 3. Asterisks indicate values significantly higher than all the control groups. (Reproduced by permission from Peter et al. Effects of transforming growth factor-beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates. J Biomed Mater Res. 2000;50:452-62.)

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Biodegradable Polymer Microparticles

Microparticles of blends (mixtures) of poly(DL-lactic-co-glycolic acid) (PLGA, Medisorb®; Alkermes; Cincinnati, Ohio) with a 50:50 lactic to glycolic acid copolymer ratio and poly(ethylene glycol) (PEG; Aldrich, Milwaukee, Wisconsin) were fabricated with use of a double-emulsion-solvent-extraction technique ([water-in-oil]-in-water) as previously described^10,23. PLGA had an average molecular weight (Mw) of 46.7 kDa and a polydispersity index (PI) of 1.73, as determined by gel permeation chromatography (GPC)¹⁷. The Mw of PEG was approximately 10.7 kDa. Recombinant human transforming growth factor-&betabeta;1 (TGF-&betabeta;1; R&D Systems, Minneapolis, Minnesota) with a Mw of 25 kDa was loaded into the PLGA/PEG microparticles.

Two types of microparticles were fabricated. The microparticles incorporating 0, 1, and 5% by weight PEG and 6 ng of TGF-&betabeta;1 per mg microparticle were used for in vitro release studies¹⁵. Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA; Sigma, St. Louis, Missouri) with a Mw of 68 kDa was co-encapsulated as a porogen at 4 g per mg microparticle. The microparticles used in cell culture studies were fabricated by co-encapsulation of TGF-&betabeta;1 and FITC-dextran with a Mw of 19.2 kDa (Sigma) into PLGA microparticles containing 5% PEG²². The theoretical loading densities were 6.0, 1.4, or 0.6 ng for TGF-&betabeta;1 and 10 g for FITC-dextran based on a 1-mg microparticle.

Size distribution of the microparticles was measured with a Coulter counter multisizer (model 0646; Coulter Electronics, Hialeah, Florida). The entrapment efficiency of the compounds was determined by normalizing the amount actually entrapped with the starting amount with use of an established solvent extraction technique¹⁰. The concentration of TGF-&betabeta;1 was analyzed by enzyme-linked immunosorbent assay (ELISA) with use of a TGF-&betabeta;1 quantikine kit (R&D Systems).

Protein Release Kinetics

The protein release kinetics from PLGA/PEG microparticles were studied under six experimental conditions¹⁵. Microparticles with an initial PEG content of 0, 1, or 5% by weight were placed in phosphate buffered saline (PBS), pH 7.4, for as long as 28 days. Additional microparticles containing 5% PEG were incubated in buffers of pH 3, 5, or 7.4. At the end of various time points, the amounts of TGF-&betabeta;1 and co-encapsulated compounds in the releasing media were analyzed by ELISA and spectrophotometry, respectively.

In Vitro Degradation of Microparticles

The degradation of PLGA/PEG microparticles (loaded with TGF-&betabeta;1 and FITC-BSA) was studied under the same conditions as in the protein release experiments during 28 days of incubation in aqueous solutions¹⁵. The PLGA Mw distribution of microparticles was determined by the gel permeation chromatography (GPC; Waters, Milford, Massachusetts) equipped with a differential refractometer (model 410; Waters). The half-life of PLGA under each experimental condition was calculated by fitting the data for Mw to an exponential function of time¹⁰. The pH of the incubation media was monitored throughout the experiment with use of a pH meter.

The morphology of degrading microparticles was assessed by scanning electron microscopy (SEM) (JEOL JSM-5300 Scanning Microscope, Boston, Massachusetts). The distribution of FITC-BSA within the microparticles was examined by fluorescence microscopy (Zeiss LSM Axiovert; Carl Zeiss, Germany).

Biodegradable Polymer Substrates

Poly(propylene fumarate) (PPF) was synthesized from fumaryl chloride and propylene glycol in the presence of potassium carbonate, a proton scavenger (all chemicals were obtained from Acros Chemical, Pittsburgh, Pennsylvania), with use of established techniques described previously¹⁹. The resulting PPF (1 g) was then mixed with N-vinyl pyrrolidone (0.33 ml) and benzoyl peroxide (0.005 g) in a cylindrical mold 1.8 cm in diameter. Cured cylinders were sectioned into 1.5-mm-thick disks. The discs were then leached out in water to remove unreacted compounds, thoroughly dried, sterilized by ethylene oxide, prewetted with sterile phosphate buffered saline, and finally utilized as substrates for cell culture.

Biological Activity of Released TGF-b1

The femurs and tibias of 6-week-old male Sprague-Dawley rats were harvested as previously described to isolate marrow stromal cells²⁴. After 10 days of proliferation in tissue-culture flasks, the marrow-derived cells were seeded at 42,000 cells/cm²onto tissue-culture polystyrene (TCPS; Fisher Scientific, Pittsburgh, Pennsylvania) and PPF substrates in TCPS wells. Complete media containing Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 20 g/ml penicillin/streptomycin/neomycin (PSN), 20 g/ml fungizone, 10^-7M dexamethasone, 10 mM Na &betabeta;-glycerol phosphate, and 50 g/ml L-ascorbic acid were added to each well. The cells were subsequently maintained in complete media for 24 hours to induce the osteoblastic phenotype of the marrow stromal cells²⁵.

The media were then replaced with fresh media (DMEM supplemented with 10% FBS, 20 g/ml PSN, and 20 g/ml) containing TGF-&betabeta;1 at 0, 0.01, 0.1, 1.0, 5.0, or 10.0 ng/ml. Alternatively, conditioned media were prepared by the incubation of PLGA/PEG blend microparticles loaded with 6 ng TGF-&betabeta;1 per mg microparticle and FITC-dextran with the primary media in transwells. The amount of microparticles used for each well (0, 0.02445, 0.2445, 2.445, 12.225, or 24.45 g) was calculated from the cumulative TGF-&betabeta;1 release curve generated previously to obtain the corresponding TGF-&betabeta;1 concentration of 0, 0.01, 0.1, 1.0, 5.0, or 10.0 g/ml, respectively, after 3 days of release. The cells were maintained in media with added TGF-&betabeta;1 or conditioned media for 3 days and subsequently assayed for alkaline phosphatase (ALP) activity. The values obtained were compared to assess the biological activity of TGF-&betabeta;1 released from the microparticles.

Osteoblastic Differentiation in Response to Released TGF-b1

Marrow-derived cells were seeded at 24,000 cells/cm² in complete media onto both TCPS and PPF substrates as described in the previous section. After 24 hours of cell seeding, PLGA/PEG microparticles were suspended in primary media and added to the top portion of transwell plates. The mass of microparticles was chosen such that a TGF-&betabeta;1 dosage of 1.0 ng/ml was achieved after 3 days of release. Cells seeded on PPF substrates and treated with blank microparticles without TGF-&betabeta;1, as well as cells seeded on PPF and TCPS and cultured in the absence of any microparticles, served as controls. Cell samples were assayed for total DNA concentration, ³H-thymidine incorporation, ALP activity, and osteocalcin production through a 21-day culture period as has been previously described²⁵.

Statistical Analysis

All data are reported as means ± standard deviations (SD) for n = 3 or 4, except for size distribution measurements where the SD was calculated on the basis of normal distribution. Single factor analysis of variance (ANOVA) was used to assess the statistical significance of the results. Scheffé’s method was employed for multiple comparison tests at significance levels of 95 and 99%.

Results

Introduction | Materials and Methods | Results | Discussion | References

PLGA/PEG Blend Microparticles

The average diameter of PLGA microparticles containing 0, 1, and 5% initial PEG was 20.0 ± 11.9, 18.8 ± 9.9, and 23.3 ± 13.7 m, respectively. The corresponding entrapment yield of TGF-&betabeta;1 was 83.4 (±13.1), 84.6 (±16.4), and 54.2 (±12.1) %, determined by normalizing the amount actually entrapped to the starting amount. The entrapment efficiency of the co-encapsulated protein FITC-BSA in the microparticles was approximately 60% for different PEG contents. The significantly lower (p < 0.05) yield of TGF-&betabeta;1 for 5% PEG may be due to the leaching of compounds with soluble PEG during microparticle fabrication. For microparticles used in cell culture studies incorporating the polysaccharide FITC-dextran, the entrapment efficiency of TGF-&betabeta;1 at different loadings was approximately 40%, resulting in actual loading densities of 2.42, 0.55, and 0.25 ng TGF-&betabeta;1 per mg microparticle.

Degradation of PLGA occurred during the microparticle fabrication process due to the contact with aqueous solutions. The degradation resulted in decreased PLGA Mw and broader Mw distribution relative to the values of the raw material. All prepared microparticles had a similar Mw of 42.8 kDa and a polydispersity index of 6.6.

In Vitro Protein Release Kinetics

Microparticle PEG content (Fig. 1-A) and medium acidity (Fig. 1-B) affected TGF-&betabeta;1 release from the microparticles. For initial PEG contents of 0, 1, and 5%, 2.9 ± 0.2, 2.7 ± 0.3, and 1.9 ± 0.3 ng of TGF-&betabeta;1 per mg microparticle was released to PBS after 2 days, respectively. While in buffers of pH 3, 5, and 7.4, 0.6 ± 0.1, 0.9 ± 0.4, and 1.9 ± 0.2 ng of TGF-&betabeta;1 per mg microparticle was released from microparticles containing 5% PEG, respectively (Fig. 1-B). At pH 7.4, the initial burst was followed by a slow linear release phase that reached a plateau. In acidic buffers, however, the initial burst was followed by a fast linear release phase (days 1-8) and then by a slower one (days 9-28). The cumulative mass of released TGF-&betabeta;1 was also increased at lower PEG content or higher buffer pH, similar to that observed for FITC-BSA. After 28 days, 3.4 ± 0.2 and 2.2 ± 0.3 ng of loaded TGF-&betabeta;1 was released from microparticles with 0 and 5% PEG in pH 7.4 PBS and 2.0 ± 0.2 and 1.3 ± 0.4 ng was released for 5% PEG in pH 7.4 and 3 buffers, respectively.

The release profiles of co-encapsulated FITC-BSA were also composed of three phases: (a) an initial burst occurring during the first 24 hours, (b) a linear steady release phase that lasted to day 8, and (c) a second longer lasting linear release phase for the rest of the time course (days 9-28). Increasing microparticle PEG content or incubation medium acidity resulted in reduced FITC-BSA release rates. By day 28, 3.8 ± 0.1 and 2.8 ± 0.3 g of loaded FITC-BSA per mg of microparticle was released for 0 and 5% PEG in pH 7.4 PBS, respectively. Approximately 2.3 ± 0.1 and 1.7 ± 0.3 g of FITC-BSA was released for 5% PEG in pH 7.4 and 3 buffers, respectively.

The cumulative mass release profiles for TGF-&betabeta;1 at different loading densities showed that 17.9 (±0.6) and 32.1 (±2.5) % of loaded TGF-&betabeta;1 was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks (Fig. 2). Incorporation of FITC-dextran allowed modulation of TGF-&betabeta;1 release profiles with a smaller burst effect as compared with FITC-BSA. A cumulative release of 52 and 33% of loaded FITC-dextran and TGF-&betabeta;1 was observed after 28 days, respectively.

In Vitro Degradation of Microparticles

The Mw of PLGA decreased continuously throughout the time course for microparticles with varied PEG contents placed in pH 7.4 PBS (Fig. 3-A). By day 28, only 35.6 (±0.5), 34.4 (± 1.3), and 29.5 (±1.5)% of the Mw at day 0 remained for microparticles with 0, 1, and 5% PEG, respectively. The half-life of PLGA for microparticles with 5% PEG was 15.9 ± 1.2 days; this was significantly lower (p < 0.05) than that for 0 and 1% PEG (20.3 ± 0.9 and 18.9 ± 0.5 days, respectively). For microparticles with 5% PEG incubated for 28 days in pH 3, 5, and 7.4 buffers (Fig. 3-B), the remaining PLGA Mw was 3.1 (±0.3), 14.0 (± 0.8), and 25.6 (±4.6)% of the day-0 value, respectively. The corresponding half-lives of PLGA were 5.5 ± 0.1, 10.9 ± 0.4, and 14.8 ± 0.4 days; these values were significantly dependent on the environmental pH (p < 0.01).

SEM micrographs of the initial PLGA/PEG microparticles revealed microspheres with smooth, non-porous surfaces (Fig. 4-A). The integrity of the microparticles with varied PEG contents was maintained after 28 days of degradation in pH 7.4 PBS (Fig. 4-B); however, significant morphological changes were noticed. The microparticles became non-spherical in shape and the surfaces were rough, revealing numerous micropores due to significant degradation (Fig. 4-B). This phenomenon was also observed for microparticles with 5% PEG incubated with acidic pH buffers. Due to faster degradation, the microparticles became irregularly shaped in pH 5 (Fig. 4-C) and completely degraded in pH 3 buffers by day 28.

Fluorescence images showed fairly uniform distribution of FITC-BSA throughout the microparticles at day 0 (Fig. 5-A). After 28 days of incubation in pH 7.4 PBS, most of the incorporated FITC-BSA was released and the remaining amount was distributed mainly at the periphery of the microparticles (Fig. 5-B). However, due to limited solubility of FITC-BSA under acidic conditions, aggregation of FITC-BSA occurred gradually in the polymer matrix, as indicated by enhanced fluorescence over the incubation period. The aggregated FITC-BSA formed a gel-like structure after significant PLGA degradation at 28 days (Fig. 5-C).

Biological Activity of Released TGF-b1

Marrow-derived stromal cells were seeded on PPF and control TCPS surfaces and exposed to different concentrations of TGF-&betabeta;1 in the medium. The ALP activity of the cells exhibited a dose response to TGF-&betabeta;1 (Fig. 6-A). The values were normalized to that for control TCPS in the absence of TGF-&betabeta;1: 0.14 (±0.03) 10^-7 mole/cell/min. After 3 days, the cells showed the highest ALP activity in medium with a TGF-&betabeta;1 concentration of 1.0 ng/ml, reaching 8.5 ± 2.0 and 5.9 ± 1.4 times that of the control value on PPF and TCPS substrates, respectively. The TGF-&betabeta;1 dosage of 1.0 ng/ml in the medium was therefore considered optimal for this study.

For marrow-derived cells cultured in media conditioned with TGF-&betabeta;1 released from PLGA/PEG microparticles with estimated concentrations similar to those directly added, the ALP activity of the cells responded to TGF-&betabeta;1 in a similar concentration-dependent manner (Fig. 6-B). The values were also normalized to that for control TCPS in the absence of TGF-&betabeta;1: 0.34 (±0.09) 10^-7 mole/cell/min. After 3 days, the ALP activity was 6.48 ± 1.90 and 7.00 ± 2.03 times that of the control value for PPF and TCPS substrates, respectively. These values were comparable with those obtained for added TGF-&betabeta;1, indicating the retention of bioactivity of TGF-&betabeta;1 during microparticle fabrication and after release.

Cell Function and Osteoblastic Differentiation in Response to Released TGF-b1

The effects of TGF-&betabeta;1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and function were assessed during a 21-day culture period. Cellular proliferation was determined by measuring the incorporation of ³H-thymidine into the DNA of replicating cells. The proliferation was most rapid during the first 24 hours of culture, with mean values ranging from 0.41 to 0.47 counts/min/cell (Fig. 7-A). The proliferative activity remained relatively high through day 4, followed by decreased levels of ³H-thymidine uptake for the rest of the time course. No statistical difference between the ³H-thymidine incorporation of any sample set was observed. These results coincided with the total cell numbers obtained from the DNA assay (Fig. 7-B). The cell numbers increased rapidly for all sample sets through day 7, reaching a plateau afterward. The sample sets exposed to released TGF-&betabeta;1 had significantly higher cell counts after day 7. By day 21, the total number of cells cultured on PPF in the presence of TGF-&betabeta;1 had reached 138,700 ± 3,300 cells/cm², while those for control sample sets TCPS, PPF, and blank microparticles were 123,700 ± 2,300, 114,300 ± 3,900, and 119,600 ± 6,100 cells/cm², respectively.

Cell function was monitored by determining the expression of two markers of the osteoblastic phenotype: ALP activity and osteocalcin production. At day 7, all sample sets had a moderate level of ALP activity, with no difference in activity between sample sets (Fig. 7-C). However, at days 14 and 21, the ALP activity was significantly higher (p < 0.05) for the cells maintained in the presence of TGF-&betabeta;1 than for all control conditions. The ALP activity for this sample set at day 21 was 22.8 (±1.5) 10^-7 mole/min/cell, significantly higher than the next highest value of 16.3 (± 2.9) 10^-7 mole/min/cell for the cells cultured on PPF with blank microparticles. The effect of TGF-&betabeta;1 release was similar for osteocalcin production. The cells exposed to TGF-&betabeta;1 had a significantly higher (p < 0.05) level of osteocalcin released into the medium than those maintained in the absence of TGF-&betabeta;1 (Fig. 7-D), reaching 15.9 (±1.5) 10^-6ng/cell at this time point. The control cultures had values of 13.0 (± 1.0) 10^-6 ng/cell for TCPS substrates, 12.5 (±1.6) 10^-6 ng/cell for PPF substrates, and 12.2 (±1.2) 3 10^-6 ng/cell for PPF cultures exposed to blank microparticles.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

PLGA/PEG blend microparticles loaded with TGF-&betabeta;1 were fabricated by an established double-emulsion-solvent-extraction technique¹⁵. The microparticles were spherical with smooth, non-porous surfaces and had an average size of about 20 m. The actual weight percent of PEG incorporated into the microparticles has been previously determined as 0.49 and 1.49% for an initial PEG content of 1 and 5%, respectively¹⁰. Both TGF-&betabeta;1 and the co-encapsulated molecule (FITC-BSA or FITC-dextran) were entrapped at high efficiencies. The actual values were likely to be higher due to an underestimation of the entrapment yield caused by compound adsorption at the organic-aqueous interface during the extraction process. Moreover, FITC-BSA or FITC-dextran (and probably TGF-&betabeta;1) was dispersed fairly uniformly throughout the microparticles (Fig. 5).

Increasing the initial PEG content from 0 to 5% resulted in the release of a lower cumulative mass of both FITC-BSA and TGF-&betabeta;1. This is in contrast to the results found in a previous study showing that the protein release rate increased with increasing PEG content¹⁰. This difference, however, is attributed to the difference in properties of incorporated compounds, such as molecule size, the compound-carrier interactions, and the actual loading density. Increasing PEG content also increased PLGA degradation, probably due to the increased surface area or greater water uptake through micropores created by the dissolution of the PEG fraction from the microparticles.

Decreasing the medium pH from 7.4 to 3 resulted in decreased protein release. Although PLGA microparticles were almost completely degraded in the pH 3 buffer, the release of both TGF-&betabeta;1 and FITC-BSA was much slower. FITC was used as a fluorescent tag for the analysis of BSA, but it had limited solubility at lower pH. This led to the aggregation of FITC-BSA in the polymer matrix, as confirmed by enhanced fluorescence after prolonged incubation. The decreased protein release at lower pH is believed to result from the aggregation of insoluble compounds. This finding is particularly interesting because heterogeneous bulk degradation of PLGA can lead to accumulation of acidic degradation products in the specimen center^17,18. The resulting lower pH may lead to changes in the properties of encapsulated molecules such as solubility, aggregation, and activity.

The TGF-&betabeta;1 release profiles could be further modulated by varying the type of co-encapsulated compounds¹⁶. A second molecule has often been used in microparticle systems as a carrier to modulate some of the characteristics of the compound of interest, including the release rate, stability, and diffusivity after release. The protein release profiles at different pH buffers suggest that the release rates of TGF-&betabeta;1 could be decreased after co-encapsulation of a less soluble molecule. In addition, when co-encapsulated with FITC-dextran, 54% of the total released TGF-&betabeta;1 was freed from PLGA/PEG microparticles after 24 hours, in contrast to a value of 75% in a previous study using FITC-BSA^15,22. The release kinetics of TGF-&betabeta;1 often differ from those of the co-encapsulated compound, due to the differences in structure, size, charge, solubility, loading density, and interactions with the polymer matrix of the two molecules. Altering the loading density of TGF-&betabeta;1 allowed further modulation of the amount of TGF-&betabeta;1 released.

Many important parameters have been identified that affect the protein release kinetics from biodegradable microparticles, including the properties of the protein, co-encapsulated molecules, the polymer matrix, and their complex interactions, as well as the microparticle fabrication process and the environmental conditions^4,16,26-28. A theoretical model for protein release from biodegradable microparticles has been established²⁷. In the present study, FITC-BSA was released in a triphasic fashion. The initial burst was due to the desorption of proteins at the surface. Polymer hydration and protein diffusion led to a linear release phase. Subsequent solubilization and release of low-Mw degradation products from the microparticles resulted in polymer mass loss and a second linear protein release phase. TGF-&betabeta;1 exhibited biphasic release profiles in pH 7.4 PBS. Although the initial burst effect appeared more significant, the percentage of the cumulative release (normalized to actual loading) was about 70% after 28 days, suggesting a longer time course for complete release.

TGF-&betabeta;1 incorporated into PLGA/PEG blend microparticles was shown to retain its activity during the microparticle fabrication process and was released not only in a controlled fashion but also in a bioactive form. Marrow stromal cells showed similar dose responses when cultured in the presence of various concentrations of TGF-&betabeta;1 prepared by the addition of known amounts of TGF-&betabeta;1 or TGF-&betabeta;1-loaded PLGA/PEG microparticles. To eliminate batch-to-batch variations, the values for ALP activity were normalized to those for control TCPS in the absence of TGF-&betabeta;1. An optimal TGF-&betabeta;1 dosage of 1.0 ng/ml in the medium was determined for maximal ALP activity.

The released TGF-&betabeta;1 from PLGA/PEG microparticles was found to enhance marrow stromal cell proliferation and differentiation over a 21-day time frame when cultured on biodegradable PPF substrates. By day 21, the total cell number, ALP activity, and osteocalcin production were significantly higher for cells maintained in the presence of released TGF-&betabeta;1 than for all other experimental conditions. Approximately 56 and 22% increases of expression over the TCPS controls were observed for ALP and osteocalcin levels, respectively.

The effects of released growth factors on cell function and tissue formation have been investigated in vitro and in vivo. TGF-&betabeta;1 has been shown to increase bone formation in critical-sized defect models²⁹. Bone morphogenetic proteins (BMPs), which also belong to the TGF-&betabeta; superfamily, induced the osteoblastic phenotype of marrow stromal cells in vitro when released from porous PLGA microparticles³⁰. Promotion of new bone formation by BMPs has also been observed in various bone defects^4,31-34. However, the optimal conditions for TGF-&betabeta; release during bone regeneration at a skeletal defect site have yet to be defined. Key questions such as optimal dosages and their temporal and spatial presentation need to be addressed before these delivery systems can be used most effectively in clinical applications. Nevertheless, the PLGA/PEG delivery system developed in this study, in combination with a novel injectable biodegradable formulation based on PPF, is promising for bone tissue induction in vivo.

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Topics

buffers ; fluorescein-5-isothiocyanate ; osteoblasts ; polymers ; ethylene glycol ; cell-derived microparticles

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Lichun Lu, Michael J. Yaszemski, Antonios G. Mikos; TGF-β1 Release from Biodegradable Polymer Microparticles: Its Effects on Marrow Stromal Osteoblast Function. The Journal of Bone & Joint Surgery. 2001 Apr;83(1_suppl_2):S82-S92.

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