Growth Factor Delivery from Tissue Engineering Matrices: Inducing

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Growth Factor Delivery from Tissue Engineering Matrices: Inducing Angiogenesis to Enhance Transplanted Cell Engraftment 1

Martin C. Peters and David J . Mooney1-4 2

Departments of1BiomedicalEngineering, Biologic and Materials Sciences, and Chemical Engineering, University of Michigan, Ann Arbor, MI 48109 3

Tissue engineering has been developed to address the increasing demand for replacement tissues and organs. It may be possible to guide the formation o f new functional tissues by using three-dimensional biodegradable porous matrices for cell transplantation. One significant challenge facing tissue engineering is the lack of nutrients i n i t i a l l y a v a i l a b l e to transplanted c e l l s . A potential approach to address this nutrient transport limitation is to encourage the r a p i d f o r m a t i o n o f a f u n c t i o n a l vasculature w i t h i n the transplanted matrix. Angiogenesis (blood vessel formation) can be promoted v i a the delivery o f protein growth factors in the area of transplanted cells. C o m m o n tissue engineering materials (e.g., poly lactic-co-glycolic acid (PLGA), alginate) have been used successfully to encapsulate and release angiogenic growth factors. The localized delivery of growth factors from a tissue engineering matrix m a y p r o v i d e a means to accelerate the v a s c u l a r i z a t i o n process and b r i n g the engineering o f large m e t a b o l i c a l l y active tissues one step closer to clinical application.

Engineering Organs with Cell Transplantation The field o f tissue engineering has developed due to the inadequate supply of organs and tissues for patients requiring organ/tissue replacement. For example, over 60,000 patients are currently on a waiting list to receive a new organ, and over 4000 patients die while waiting for an organ each year due to the shortage o f donated organs . A promising alternative to organ transplantation that could greatly expand 1

Corresponding author.

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Park and Mrsny; Controlled Drug Delivery ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

158 clinicians' ability to treat patients requiring new organs and tissues is the selective transplantation o f the appropriate cell type(s), and the reconstitution o f a functional 2

tissue mass from these transplanted c e l l s . Thus, cells from a single donated organ could be utilized to treat multiple patients, or a small biopsy from a living donor could yield a sufficient number o f cells to engineer the necessary tissue mass. The concept of engineering organs using selective cell transplantation has recently been validated in animal models by the demonstrations that functional new intestinal tissue can be 3

engineered and placed in-line with native intestine , and functional new bladders can 4

be similarly engineered and utilized to replace the native bladder . One promising approach to engineer new tissues using cell transplantation involves delivering cells on maeroporous matrices, which become structurally integrated with the surrounding host tissue. These matrices can be fabricated from a 5

number o f different synthetic and naturally derived materials . Synthetic polymers comprised o f g l y c o l i c and lactic acid ( P L G A ) are especially attractive for this approach, as their degradation following tissue development w i l l result in a completely natural new tissue. These polymers have been used in humans in the form 6

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o f biodegradable sutures for over 20 years - , and are considered to be biocompatible. T h e y degrade to lactic and g l y c o l i c acid, which are natural metabolites, and the degradation rate o f the polymer can be precisely controlled by varying the ratio o f 5

lactic:glycolic acid in the p o l y m e r . These polymers have been processed into matrices with a number o f physical forms, including non-woven fibrous arrays and 8

foams, utilizing a variety o f processing techniques . In this review we w i l l discuss the challenges involved in vascularization o f synthetic tissue engineering matrices, and we w i l l focus on utilizing protein growth factor delivery to induce the formation o f blood vessels. Discussions w i l l include the characteristics o f specific growth factors, materials and mechanisms for drug delivery, and strategies to apply current technology to tissue engineering.

Vascularization and Cell Survival Transplantation o f selected cell populations on biodegradable polymer matrices is an attractive approach to engineer a variety o f tissue types (e.g., liver, intestine). However, the survival o f transplanted cells is dependent on the diffusional transport o f nutrients and waste products between these cells and the surrounding host tissue. Diffusion o f oxygen is typically the limiting factor for cell survival in this situation, and it is estimated that cells more than several hundred microns from the capillaries in surrounding tissues w i l l fail to engraft or rapidly die due to oxygen depletion . Matrices utilized for cell transplantation are designed to address this mass transport issue as they contain large, interconnected pores that enhance diffusional transport o f nutrients through the matrix. In addition, the macropores are intended to promote the ingrowth o f granulation tissue from the host to provide for convective nutrient transport to the engineered tissue, and potentially allow for large tissue masses to be created. A variety o f studies indicate that blood vessels w i l l invade the maeroporous matrices utilized for cell t r a n s p l a n t a t i o n " . However, fibrovascular ingrowth into the matrix occurs at a rate less than 1 mm/day, and typically takes one to two weeks to completely penetrate even relatively thin (e.g., 3 m m thick) 9

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matrices . The net result is that a high percentage of transplanted cells die within several days following transplantation . This mass transport limitation currently limits the thickness of engineered tissues to the millimeter size scale, and this scale is clearly insufficient if one needs to replace an entire organ or a large mass of tissue. A critical challenge in the tissue engineering field is thus to develop strategies to achieve rapid and extensive vascularization of forming tissues. 14

Growth Factors It has long been appreciated that a functional vasculature is an essential component of any metabolically active tissue which has a thickness in excess of a few millimeters "'. The process of generating new microvasculature, termed angiogenesis, is a process observed physiologically in development and wound healing . A variety of tissue-inducing substances (e.g., growth factors) that promote the formation of new microvasculature have been identified and they could potentially be utilized to accelerate the vascularization of engineered tissues. These substances stimulate the appropriate cells (e.g., endothelial cells), already present in the patient's body, to migrate from the surrounding tissue, proliferate, and finally differentiate into blood vessels . By combining growth factor delivery, to induce angiogenesis, with tissue engineering matrices the engraftment of transplanted cells may be enhanced. The search for potential growth factors regulating angiogenesis has yielded numerous candidates: basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), angiogenin, prostoglandin E (PGE ), transforming factor-alpha (TGF-a), TGF-β, and vascular endothelial growth factor (VEGF) to name only a few . A l l of these molecules are able to promote angiogenesis in certain in vivo model systems but with the exception of bFGF and V E G F these agents have little or no direct mitogenic effect on vascular endothelial cells . 1

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bFGF and its receptors can be found in nearly all tissues of the body - . It is a single polypeptide of ~T6kDa (146 amino acids) and part of a family of at least seven related heparin-binding peptides . bFGF activity has been implicated in both normal and pathological processes including embryogenesis, limb regeneration, wound healing, diabetic retinopathy, and tumor angiogenesis . bFGF promotes the proliferation of a broad range of cells - (e.g., fibroblasts, keratinocytes, smooth muscle cells, endothelial cells) and is intensely angiogenic, even at picomolar concentrations, in v i v o . V E G F is a homodimer with a molecular mass of 45 kDa and appears in four distinct isoforms: VEGF, i, V E G F , VEGF, , and V E G F . V E G F is a key regulator of blood vessel formation during development and in neovascularization associated with tumors and intra-ocular disorders - . V E G F | is a secreted heparinbinding protein that acts as a potent mitogen for micro and macrovascular endothelial cells from arteries and veins but lacks mitogenic activity for other cell types . V E G F is an attractive molecule to enhance the vascularization of engineered tissues due to its endothelial cell specificity. To increase the survival of transplanted cells it will be critical to not only maximize the amount of vascularization, but also 22

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160 the mass transport capability o f the vasculature versus the metabolic needs o f the forming tissues. The other growth factors identified to date which promote angiogenesis (e.g., F G F family) also increase the proliferation o f other cell types (e.g., 16

fibroblasts) present in connective t i s s u e . The increase in mass transport obtained with these other molecules may be offset by an increased need for nutrients due to non-desirable proliferation o f host cells in the region o f the engineered tissue. Systems for the sustained and localized delivery o f b F G F and V E G F have been 30

previously d e m o n s t r a t e d "

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but only preliminary work has been done to incorporate 34

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angiogenic factors into tissue engineering m a t r i c e s ' .

G r o w t h F a c t o r Delivery Previous studies have demonstrated that it is possible to promote angiogenesis through the delivery o f peptide growth factors such as b F G F and y E G ρ 17,36-38 j ^ possible to enhance angiogenesis at the site where a new tissue is engineered by locally delivering a high concentration o f these growth factors in a sustained manner. G r o w t h factors have promise for tissue engineering but the development o f effective delivery mechanisms is essential to preserve their biological activity. B o t h b F G F and V E G F , though very potent, are rapidly degraded when injected or i n g e s t e d . G r o w t h factors are large molecular weight polypeptides which are susceptible to inactivation v i a a number o f avenues including proteolysis, aggregation, deamidation, and o x i d a t i o n - . T o avoid these pitfalls, investigators have developed a variety o f encapsulation methods to release various growth factors with controlled kinetics while maintaining the growth factor's biological activity. P L G A and alginate, two materials utilized extensively for tissue engineering, have also met with success as growth factor delivery devices. W e w i l l briefly discuss these materials as they are used in the drug delivery realm and review strategies to adapt the positive aspects o f each for tissue engineering. t m

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4 0

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PLGA Microspheres A c o m m o n approach to the delivery o f growth factors is to encapsulate the protein in biodegradable polymer microspheres. These microspheres can be implanted in the body and release entrapped growth factors as they degrade. L i k e the sutures and tissue engineering matrices mentioned above, P L G A microspheres w i l l degrade completely to natural metabolites so no retrieval o f the microspheres is necessary f o l l o w i n g complete drug release. Polymer microspheres have been manufactured from P L G A and used to encapsulate a variety o f proteins including 1 3

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E G F , human growth h o r m o n e , and V E G F

4 3

. G r o w t h factor encapsulation using a

double emulsion (water-in-oil-in-water) technique yields loading efficiencies o f approximately 5 0 % (actual/theoretical) and controlled release kinetics for several 1 3

4 2

4 3

weeks in v i t r o · · . The P L G A microsphere processing technology was improved 44

through the development o f a cryogenic non-aqueous p r o c e s s . This technique allows 9 5 % o f the protein to be loaded into the microspheres, and improved protein 45

stability is also o b s e r v e d .


161 Microspheres have been utilized in tissue engineering by combining the 13

microspheres with a cell suspension and seeding the mixture onto porous m a t r i c e s . This approach was used to deliver E G F to transplanted hepatocytes, and those matrices seeded with EGF-microspheres demonstrated approximately twice the 13

hepatocyte engraftment o f control s a m p l e s . Though effective, the use o f microspheres for tissue engineering has its limitations. When microspheres are seeded onto matrices it is difficult to control their distribution after implantation. The microspheres may produce non-uniform drug distributions throughout the matrix, or the microspheres could be displaced from the matrix in which they were seeded and cause unwanted cellular responses (e.g., fibroblast proliferation, blood vessel leakage) in the host tissue.

Alginate Beads Alginate, a natural material derived from seaweed, has also been utilized as 3 0

3 2

4 6

an encapsulation system for growth factors such as b F G F " - -

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and V E G F

3 3

.

Alginate has gentle gelling properties, and growth factor containing beads (-3 mm 33

diameter) can be formed at room temperature without the use o f organic s o l v e n t s . b F G F and V E G F can be encapsulated within alginate beads with a protein loading efficiency o f 30-70% depending on the concentration o f the alginate solution and the 31

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presence o f stabilizing molecules (e.g., h e p a r i n ) - . Alginate beads w i l l release the incorporated growth factor in a controlled fashion for several weeks in vitro when maintained at physiologic conditions. N o t only does the released growth factor retain its biologic activity, but some observations suggest that the negatively charged alginate interacts with the positively charged growth factor to stabilize the molecule 3 2

3 3

and enhance its activity in v i t r o ' . Alginate shows a promising interaction with growth factors and is being 48

studied for tissue engineering a p p l i c a t i o n s . However, the alginate bead system for growth factor delivery has several disadvantages that make it non-ideal for tissue engineering. Alginate, in its native form, does not undergo b i o d é g r a d a t i o n and the high molecular weight alginate typically utilized may be too large (> 150 k D a ) to pass through the kidneys. In addition, when the release o f growth factors from alginate beads is monitored, for extended times (>70 days), it is apparent that 20-30% o f the 31

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growth factor is retained in the b e a d - . This remaining growth factor may aggregate within the hydrated bead, and in this case would not be released in a functional state. Recently novel alginate-derived polymers that degrade via 49

hydrolysis to low molecular weight products have been d e v e l o p e d . These polymers eliminate this problem, and could find great utility in growth factor delivery.

Re/ease from PLGA Tissue Engineering Matrices A more elegant approach to using growth factor delivery for tissue 34

engineering is to incorporate growth factors directly into the porous m a t r i x . A gasfoaming/particulate leaching method to fabricate tissue engineering matrices with controlled porosity, without the use o f organic solvents or high temperatures has been developed using the same P L G A polymer used to fabricate m i c r o s p h e r e s

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(Fig. 1 ).

This represents a critical advance, as all other processing techniques utilized to date to


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Figure 1. Scanning electron micrograph showing a cross-section of a 85:15 PLGA matrix containing 5% (w/w) sodium alginate formed by gas-foaming/particulate leaching process (500 pm size bar shown on photograph). 8

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fabricate polymer matrices involve organic solvents and/or high t e m p e r a t u r e s - "

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w h i c h could potentially damage incorporated proteins during processing. Sheridan et al. demonstrated that lyophilized V E G F could be incorporated into the bulk o f gasfoamed/particulate leached matrices (Fig. 2).

Figure 2. Process for fabricating PLGA matrices with incorporated growth factor. Lyophilized growth factor, (black circles), PLGA particles (shaded circles), and NaCl particles (gray squares) are mixed and compression molded to create a disk 3 mm in thickness. The compressed pellet is equilibrated with high-pressure carbon dioxide (800 psi), and then the gas pressure is rapidly re/eased to ambient. The PLGA particles expand into the space between the NaCl particles (indicated by hatched


163 bars), and fuse. This expansion is caused by the formation of gas pores within the particles. The NaCl and growth factor are entrapped within the continuous PLGA phase. The NaCl can subsequently be removed by leaching with water (leaving void spaces) to yield maeroporous matrices with entrapped growth factor. The V E G F was released for over 2 months in vitro and found to retain over 9 0 % o f its bioactivity for the first 2 weeks ( F i g . 3). However, the final V E G F loading was only 2 8 % o f the starting material due to the particulate-leaching step o f matrix processing. In an effort to increase growth factor loading, alginate was added to the 35

matrices prior to f o a m i n g . The addition o f alginate increased the incorporation efficiency to approximately 6 0 % while still providing controlled release kinetics for a variety o f growth factors ( F i g . 4).

0 ng VEGF

5 ng VEGF

20 ng VEGF

Day 0-2

Day 2-7

Day 7-1 4

Figure 3. Biological activity of VEGF incorporated into 75:25 PLGA matrices. The bioactivity was determined by measuring the growth stimulation of cultured endothelial cells compared to endothelial celts grown in the presence of varying 34

VEGF concentrations. Values represent mean and standard deviation (n= 3) divided by the average growth observed in control cultures.

G r o w t h factor delivery from P L G A tissue engineering scaffolds provides several potential advantages over using different scaffolds for cell and drug delivery. The scaffold can be designed to degrade at a variety o f rates, and releases growth factor throughout its life. The scaffold provides uniform growth factor concentrations in the tissue forming within the scaffold, and eliminates the need to introduce a second drug carrying material into the matrix. In addition, excipients such as alginate can easily be added to the sponge formulation prior to foaming to stabilize the growth factor and increase incorporation.


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Conclusions Engineering new tissues using cell transplantation on polymer matrices is an exciting approach to create a variety o f organs and tissues. However, the rapid development o f an appropriate vascular network throughout the matrix f o l l o w i n g cell transplantation is a critical, and currently unachieved, requirement for this approach to be applied to large organs and tissues. The localized delivery o f angiogenic factors may provide a means to accelerate the vascularization process and bring the engineering o f large metabolically active tissues (e.g., liver, kidney, and muscle) one step closer to clinical application. The demonstration that V E G F can be delivered from porous tissue engineering scaffolds is a significant contribution with regard to developing transplant matrices that interact with cells by altering their soluble microenvironment. These growth factor releasing scaffolds must next be extensively evaluated in v i v o to measure their ability to elicit physiologic responses. The development o f this technology can hopefully help usher a new generation o f tissue engineering matrices. These matrices could potentially interact with transplanted and native cells by presenting a host o f spatially and temporally regulated signals to promote controlled tissue development (e.g., vascularization, enervation) and maturation (e.g., extracellular matrix remodeling, large blood vessel formation).


165 Acknowledgements T h e authors w o u l d like to acknowledge the support o f the N S F (Grant N o . B E S - 9 5 0 1 3 7 6 ) , N I H (Grant N o . R 2 9 D K 5 0 7 1 5 ) , and Reprogenesis Inc. M . C . P was supported by a fellowship from the Whitaker Foundation.

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Growth Factor Delivery from Tissue Engineering Matrices: Inducing

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