Ana Jaklenec - Academia.edu (original) (raw)

Papers by Ana Jaklenec

ACS applied materials & interfaces, Jan 29, 2015

A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer ... more A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer films (HT-LbL) to facilitate rapid film generation, systematic film characterization, and rational investigations into their interactions with cells is described. Key parameters, such as polyelectrolyte adsorption time and polyelectrolyte deposition pH, were used to modulate LbL film growth to create LbL films of distinct thicknesses using the widely utilized polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). We highlight how HT-LbL can be used to rapidly characterize film-forming parameters and robustly create linearly growing films of various molecular architectures. Film thickness and growth rates of HT-LbL films were shown to increase as a function of adsorption time. Subsequently, we investigated the role that polyelectrolyte solution pH (ranging from 2.5 to 9) has in forming molecularly distinct films of weak polyelectrolytes and report the effect t...

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-b 1 ) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-b 1 from modular designed poly(L,D-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-b 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-b 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-b 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-b 1 released continuously, TGF-b 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-b 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-b 1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-β1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-β1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-β1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-β1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-β1 released continuously, TGF-β1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-β1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-β1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, Jan 31, 2008

Biodegradable scaffolds play an important role in tissue engineering by providing physical and bi... more Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(L,D-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3758.0 mm) rather than smaller (11.15711.08 mm) microspheres, generated pores on the order of 200 mm as compared to 20 mm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4 h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications. r

Journal of Controlled Release, 2015

Currently, vaccination is the most efficient and cost-effective medical treatment for infectious ... more Currently, vaccination is the most efficient and cost-effective medical treatment for infectious diseases; however, each year 10 million infants remain underimmunized due to current vaccination schedules that require multiple doses to be administered across months or years. These dosing regimens are especially challenging in the developing world where limited healthcare access poses a major logistical barrier to immunization. Over the past four decades, researchers have attempted to overcome this issue by developing single-administration vaccines based on controlled-release antigen delivery systems. These systems can be administered once, but release antigen over an extended period of time to elicit both a primary and secondary immune response resulting in antigen-specific immunological memory. Unfortunately, unlike controlled release systems for drugs, single-administration vaccines have yet to be commercialized due to poor antigen stability and difficulty in obtaining unconventional release kinetics. This review discusses the current state of single-administration vaccination, challenges delaying the development of these vaccines, and potential strategies for overcoming these challenges.

Tissue engineering. Part A, 2008

This report draws upon data from a variety of sources to provide a detailed estimate of the curre... more This report draws upon data from a variety of sources to provide a detailed estimate of the current scope of private sector development and commercial activity in the aggregate field comprising tissue engineering, regenerative medicine, and stem cell therapeutics. Economic activity has grown a remarkable fivefold in the past 5 years. As of mid-2007 approximately 50 firms or business units with over 3000 employees offered commercial tissue-regenerative products or services with generally profitable annual sales in excess of $1.3 billion. Well over a million patients have been treated with these products. In addition, 110 development-stage companies with over 55 products in FDA-level clinical trials and other preclinical stages employed approximately 2500 scientists or support personnel and spent 850 million development dollars in 2007. These totals represent a remarkable recovery from the downturn of 2000-2002, at which time tissue engineering was in shambles because of disappointing...

Tissue Engineering Part B: Reviews, 2012

This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and... more This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and stem cell industry. Data are reported through mid 2011 showing an almost three-fold growth in commercial sales over the past 4 years. In addition, the number of companies selling products or offering services has increased over two-fold to 106, and they are generating a remarkable 3.5billioninsales.Overall,theTEandstemcellsectorisspending3.5 billion in sales. Overall, the TE and stem cell sector is spending 3.5billioninsales.Overall,theTEandstemcellsectorisspending3.6 billion and employing almost 14,000 employees. These data suggest the TE and stem cell industry has stabilized and is on a path pointing toward continued success.

Journal of Microencapsulation, 2006

A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was car... more A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was carried out using the formation of a w/o/o emulsion followed by solvent removal. Various factors were studied, including composition of the suspension medium and the relative amounts of aqueous phase containing protein to polymer solution. High yields of microsphere fabrication were achieved by using silicon oil containing methylene chloride as a suspension medium instead of pure silicon oil, with minimal loss of polymer and protein drug (<2%). The amount of aqueous phase influenced the process and successful encapsulation was obtained if the volume ratios of aqueous phase to polymer solution were less than 5% (v/v) at a wide range of polymer concentration (2-15% g ml-1). Protein encapsulation by this w/o/o emulsion and solvent removal method has a high yield of microsphere fabrication and protein encapsulation (98%). In addition, it provides an easy way to control the release rate of protein encapsulated in microspheres by modulating their porosity in fabrication process.

IET Synthetic Biology, 2007

In 2000, Gardner and Collins reported the construction of a fundamental genetic regulatory device... more In 2000, Gardner and Collins reported the construction of a fundamental genetic regulatory device, the bi-stable toggle switch, in E. coli. We report here our work on a natural extension of this powerful device, a tri-stable genetic toggle switch capable of switching among three stable states. Like the bi-stable switch, the tri-stable switch consists of repressible promoters that produce inhibitory proteins and requires only a transient pulse of chemical inducer to switch among stable states. Our proof-of-principal construct is designed to control the expression of three different fluorescent reporters using the pBad/AraC, pLacI/LacI, and pTetR/TetR systems; though a tri-stable switch can theoretically be constructed from any three repressible promoters that satisfy a certain mathematical relationship. We have modelled the system extensively, creating both a simple continuous deterministic model based on the work of and a more complex discrete stochastic model based on the work of Isaacs (Isaacs, 2003). The tri-stable switch, designed, modelled, and partially constructed as an iGEM 2006 project at Brown University, is to be composed entirely of Biobricked parts from the Registry of Standard Biological Parts. In addition to providing support for the iGEM hypothesis, the tri-stable toggle switch has implications for biotechnology and gene therapy.

BMC Systems Biology, 2007

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-b 1 ) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-b 1 from modular designed poly(L,D-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-b 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-b 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-b 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-b 1 released continuously, TGF-b 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-b 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-b 1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, 2008

Biodegradable scaffolds play an important role in tissue engineering by providing physical and bi... more Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(L,D-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3758.0 mm) rather than smaller (11.15711.08 mm) microspheres, generated pores on the order of 200 mm as compared to 20 mm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4 h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications. r

Journal of Pharmaceutical Sciences, 2003

A central issue in controlled delivery of therapeutics from biodegradable microspheres is the imm... more A central issue in controlled delivery of therapeutics from biodegradable microspheres is the immediate burst of drug release upon injection. This burst is often observed with microsphere systems made by the double emulsion (w/o/w) technique, and may be prevented by improving the drug distribution throughout the polymer matrix. To this end, protein and polymer (poly-lactide-co-glycolide or PLGA) were dissolved within the same solvent system, and micron-sized microspheres were created from this solution by spontaneous emulsification. Improved protein loading was achieved by ion-pairing the protein with charged surfactants to increase solubility in the single-phase solvent system. Both in vitro and in vivo results showed a much diminished burst: compared to microspheres made by double emulsion, it was reduced over 10-fold. ß

ACS applied materials & interfaces, Jan 29, 2015

A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer ... more A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer films (HT-LbL) to facilitate rapid film generation, systematic film characterization, and rational investigations into their interactions with cells is described. Key parameters, such as polyelectrolyte adsorption time and polyelectrolyte deposition pH, were used to modulate LbL film growth to create LbL films of distinct thicknesses using the widely utilized polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). We highlight how HT-LbL can be used to rapidly characterize film-forming parameters and robustly create linearly growing films of various molecular architectures. Film thickness and growth rates of HT-LbL films were shown to increase as a function of adsorption time. Subsequently, we investigated the role that polyelectrolyte solution pH (ranging from 2.5 to 9) has in forming molecularly distinct films of weak polyelectrolytes and report the effect t...

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-b 1 ) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-b 1 from modular designed poly(L,D-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-b 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-b 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-b 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-b 1 released continuously, TGF-b 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-b 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-b 1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-β1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-β1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-β1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-β1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-β1 released continuously, TGF-β1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-β1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-β1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, Jan 31, 2008

Biodegradable scaffolds play an important role in tissue engineering by providing physical and bi... more Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(L,D-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3758.0 mm) rather than smaller (11.15711.08 mm) microspheres, generated pores on the order of 200 mm as compared to 20 mm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4 h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications. r

Journal of Controlled Release, 2015

Currently, vaccination is the most efficient and cost-effective medical treatment for infectious ... more Currently, vaccination is the most efficient and cost-effective medical treatment for infectious diseases; however, each year 10 million infants remain underimmunized due to current vaccination schedules that require multiple doses to be administered across months or years. These dosing regimens are especially challenging in the developing world where limited healthcare access poses a major logistical barrier to immunization. Over the past four decades, researchers have attempted to overcome this issue by developing single-administration vaccines based on controlled-release antigen delivery systems. These systems can be administered once, but release antigen over an extended period of time to elicit both a primary and secondary immune response resulting in antigen-specific immunological memory. Unfortunately, unlike controlled release systems for drugs, single-administration vaccines have yet to be commercialized due to poor antigen stability and difficulty in obtaining unconventional release kinetics. This review discusses the current state of single-administration vaccination, challenges delaying the development of these vaccines, and potential strategies for overcoming these challenges.

Tissue engineering. Part A, 2008

This report draws upon data from a variety of sources to provide a detailed estimate of the curre... more This report draws upon data from a variety of sources to provide a detailed estimate of the current scope of private sector development and commercial activity in the aggregate field comprising tissue engineering, regenerative medicine, and stem cell therapeutics. Economic activity has grown a remarkable fivefold in the past 5 years. As of mid-2007 approximately 50 firms or business units with over 3000 employees offered commercial tissue-regenerative products or services with generally profitable annual sales in excess of $1.3 billion. Well over a million patients have been treated with these products. In addition, 110 development-stage companies with over 55 products in FDA-level clinical trials and other preclinical stages employed approximately 2500 scientists or support personnel and spent 850 million development dollars in 2007. These totals represent a remarkable recovery from the downturn of 2000-2002, at which time tissue engineering was in shambles because of disappointing...

Tissue Engineering Part B: Reviews, 2012

This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and... more This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and stem cell industry. Data are reported through mid 2011 showing an almost three-fold growth in commercial sales over the past 4 years. In addition, the number of companies selling products or offering services has increased over two-fold to 106, and they are generating a remarkable 3.5billioninsales.Overall,theTEandstemcellsectorisspending3.5 billion in sales. Overall, the TE and stem cell sector is spending 3.5billioninsales.Overall,theTEandstemcellsectorisspending3.6 billion and employing almost 14,000 employees. These data suggest the TE and stem cell industry has stabilized and is on a path pointing toward continued success.

Journal of Microencapsulation, 2006

A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was car... more A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was carried out using the formation of a w/o/o emulsion followed by solvent removal. Various factors were studied, including composition of the suspension medium and the relative amounts of aqueous phase containing protein to polymer solution. High yields of microsphere fabrication were achieved by using silicon oil containing methylene chloride as a suspension medium instead of pure silicon oil, with minimal loss of polymer and protein drug (<2%). The amount of aqueous phase influenced the process and successful encapsulation was obtained if the volume ratios of aqueous phase to polymer solution were less than 5% (v/v) at a wide range of polymer concentration (2-15% g ml-1). Protein encapsulation by this w/o/o emulsion and solvent removal method has a high yield of microsphere fabrication and protein encapsulation (98%). In addition, it provides an easy way to control the release rate of protein encapsulated in microspheres by modulating their porosity in fabrication process.

IET Synthetic Biology, 2007

In 2000, Gardner and Collins reported the construction of a fundamental genetic regulatory device... more In 2000, Gardner and Collins reported the construction of a fundamental genetic regulatory device, the bi-stable toggle switch, in E. coli. We report here our work on a natural extension of this powerful device, a tri-stable genetic toggle switch capable of switching among three stable states. Like the bi-stable switch, the tri-stable switch consists of repressible promoters that produce inhibitory proteins and requires only a transient pulse of chemical inducer to switch among stable states. Our proof-of-principal construct is designed to control the expression of three different fluorescent reporters using the pBad/AraC, pLacI/LacI, and pTetR/TetR systems; though a tri-stable switch can theoretically be constructed from any three repressible promoters that satisfy a certain mathematical relationship. We have modelled the system extensively, creating both a simple continuous deterministic model based on the work of and a more complex discrete stochastic model based on the work of Isaacs (Isaacs, 2003). The tri-stable switch, designed, modelled, and partially constructed as an iGEM 2006 project at Brown University, is to be composed entirely of Biobricked parts from the Registry of Standard Biological Parts. In addition to providing support for the iGEM hypothesis, the tri-stable toggle switch has implications for biotechnology and gene therapy.

BMC Systems Biology, 2007

Biomaterials, 2008

Growth factors have become an important component for tissue engineering and regenerative medicin... more Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-b 1 ) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-b 1 from modular designed poly(L,D-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-b 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-b 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-b 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-b 1 released continuously, TGF-b 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-b 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-b 1 sequentially makes them very useful for cartilage tissue engineering applications.

Biomaterials, 2008

Biodegradable scaffolds play an important role in tissue engineering by providing physical and bi... more Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(L,D-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3758.0 mm) rather than smaller (11.15711.08 mm) microspheres, generated pores on the order of 200 mm as compared to 20 mm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4 h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications. r

Journal of Pharmaceutical Sciences, 2003

A central issue in controlled delivery of therapeutics from biodegradable microspheres is the imm... more A central issue in controlled delivery of therapeutics from biodegradable microspheres is the immediate burst of drug release upon injection. This burst is often observed with microsphere systems made by the double emulsion (w/o/w) technique, and may be prevented by improving the drug distribution throughout the polymer matrix. To this end, protein and polymer (poly-lactide-co-glycolide or PLGA) were dissolved within the same solvent system, and micron-sized microspheres were created from this solution by spontaneous emulsification. Improved protein loading was achieved by ion-pairing the protein with charged surfactants to increase solubility in the single-phase solvent system. Both in vitro and in vivo results showed a much diminished burst: compared to microspheres made by double emulsion, it was reduced over 10-fold. ß