Eben Alsberg

Cell-interactive Alginate Hydrogels for Bone Tissue Engineering

There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGDcontaining peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.

Engineering growing tissues

Regenerating or engineering new tissues and organs may one day allow routine replacement of lost or failing tissues and organs. However, these engineered tissues must not only grow to fill a defect and integrate with the host tissue, but often they must also grow in concert with the changing needs of the body over time. We hypothesized that tissues capable of growing with time could be engineered by supplying growth stimulus signals to cells from the biomaterial used for cell transplantation. In this study, chondrocytes and osteoblasts were cotransplanted on hydrogels modified with an RGD-containing peptide sequence to promote cell multiplication. New bone tissue was formed that grew in mass and cellularity by endochondral ossification in a manner similar to normal long-bone growth. Transplanted cells organized into structures that morphologically and functionally resembled growth plates. These engineered tissues could find utility in treating diseases and injuries of the growth plate, testing the effect of experimental drugs on growth-plate function and development, and investigating the biology of long-bone growth. Furthermore, this concept of promoting the growth of engineered tissues could find great utility in engineering numerous tissue types by way of the transplantation of a small number of precursor cells.

Degradation of Partially Oxidized Alginate and Its Potential Application for Tissue Engineering

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon‐carbon bond of the cis‐diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (∼5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross‐linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate‐derived hydrogels greatly improved cartilage‐like tissue formation in vivo, as compared to alginate hydrogels.

Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.

Photocrosslinked and biodegradable alginate hydrogels were engineered for biomedical applications. Photocrosslinkable alginate macromers were prepared by reacting sodium alginate and 2-aminoethyl methacrylate in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide. Methacrylated alginates were photocrosslinked using ultraviolet light with 0.05% photoinitiator. The swelling behavior, elastic moduli, and degradation rates of photocrosslinked alginate hydrogels were quantified and could be controlled by varying the degree of alginate methacrylation. The methacrylated alginate macromer and photocrosslinked alginate hydrogels exhibited low cytotoxicity when cultured with primary bovine chondrocytes. In addition, chondrocytes encapsulated in these hydrogels remained viable and metabolically active as demonstrated by Live/Dead cell staining and MTS assay. These photocrosslinked alginate hydrogels, with tailorable mechanical properties and degradation rates, may find great utility as therapeutic materials in regenerative medicine and bioactive factor delivery.

Regulating Bone Formation via Controlled Scaffold Degradation

It is widely assumed that coupling the degradation rate of polymers used as cell transplantation carriers to the growth rate of the developing tissue will improve its quantity or quality. To test this hypothesis, we developed alginate hydrogels with a range of degradation rates by gamma-irradiating high-molecular-weight alginate to yield polymers of various molecular weights and structures. Decreasing the size of the polymer chains increased the degradation rate in vivo, as measured by implant retrieval rates, masses, and elastic moduli. Rapidly and slowly degrading alginates, covalently modified with RGD-containing peptides to control cell behavior, were then used to investigate the effect of biodegradation rate on bone tissue development in vivo. The more rapidly degrading gels led to dramatic increases in the extent and quality of bone formation. These results indicate that biomaterial degradability is a critical design criterion for achieving optimal tissue regeneration with cell transplantation.

Control of Basement Membrane Remodeling and Epithelial Branching Morphogenesis in Embryonic Lung by Rho and Cytoskeletal Tension

Local alterations in the mechanical compliance of the basement membrane that alter the level of isometric tension in the cell have been postulated to influence tissue morphogenesis. To explore whether cell tension contributes to tissue pattern formation in vivo, we modulated cytoskeletal force generation in embryonic mouse lung (embryonic days 12–14) rudiments using inhibitors of Rho‐associated kinase (ROCK), myosin light chain kinase, myosin ATPase, and microfilament integrity, or a Rho stimulator (cytotoxic necrotizing factor‐1). Tension inhibition resulted in loss of normal differentials in basement membrane thickness, inhibition of new terminal bud formation, and disorganization of epithelial growth patterns as well as disruption of capillary blood vessels. In contrast, increasing cell tension through Rho activation, as confirmed by quantitation of myosin light chain phosphorylation and immunohistocytochemical analysis of actin organization, accelerated lung branching and increase capillary elongation. These data suggest that changes in cytoskeletal tension mediated by Rho signaling through ROCK may play an important role in the establishment of the spatial differentials in cell growth and extracellular matrix remodeling that drive embryonic lung development. Developmental Dynamics 232:268–281, 2005.

Tissue-Engineered Small Intestine Improves Recovery After Massive Small Bowel Resection

Objective:Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR). Summary Background Data:Short bowel syndrome is a morbid product of massive small bowel resection. We report the first replacement of a vital organ by tissue engineering with TESI after MSBR. Methods:Ten male Lewis rats underwent TESI implantation with green fluorescent protein (GFP)-marked cells (TESI+, n = 5) or sham laparotomy (TESI−, n = 5) followed by MSBR. Side-to-side anastomosis of TESI to proximal small intestine was performed or omitted. TESIØ animals underwent implantation of engineered intestine with no further surgery. Weights were measured QOD until day 40. Transit times were measured. DNA assay was performed with computer morphometry. Northern blots of RNA were probed for intestinal alkaline phosphatase (IAP) and villin. Hematoxylin and eosin, S100, and smooth muscle actin immunohistochemistry were performed. Blood was collected at sacrifice. Results:All 10 rats initially lost then regained weight. The initial rate of weight loss was higher in TESI+ versus TESI−, but the nadir was reached a week earlier with more rapid weight gain subsequently to 98% preoperative weight on day 40 in animals with engineered intestine versus 76% (P < 0.03). Serum B12 was higher at 439 pg/mL versus 195.4 pg/mL. IAP mRNA appeared greater in TESI+ than TESIØ, with constant villin levels. Histology revealed appropriate architecture including nerve. GFP labeling persisted. Conclusions:Anastomosis of TESI significantly improved postoperative weight and B12 absorption after MSBR. IAP, a marker of differentiation in intestinal epithelium, is present in TESI, and GFP labeling was accomplished.

Craniofacial tissue engineering

There is substantial need for the replacement of tissues in the craniofacial complex due to congenital defects, disease, and injury. The field of tissue engineering, through the application of engineering and biological principles, has the potential to create functional replacements for damaged or pathologic tissues. Three main approaches to tissue engineering have been pursued: conduction, induction by bioactive factors, and cell transplantation. These approaches will be reviewed as they have been applied to key tissues in the craniofacial region. While many obstacles must still be overcome prior to the successful clinical restoration of tissues such as skeletal muscle and the salivary glands, significant progress has been achieved in the development of several tissue equivalents, including skin, bone, and cartilage. The combined technologies of gene therapy and drug delivery with cell transplantation will continue to increase treatment options for craniofacial cosmetic and functional restoration.

Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels

Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications.

Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering

Degradable and injectable hydrogels may be ideal for bone-tissue engineering, especially in the craniofacial region because of the ease of access for injection. Alginate hydrogels potentially could be used as injectable cell delivery vehicles, but they exhibit a limited range of mechanical properties and uncontrollable disintegration time. Therefore we synthesized new hydrogels, composed of poly(aldehyde guluronate) (PAG) and adipic acid dihydrazide, that have a wide range of mechanical stiffness and controllable degradation rate. MC3T3-E1 cells adhered and multiplied on PAG hydrogels in vitro. When primary rat calvarial osteoblasts were mixed with PAG hydrogels and subcutaneously injected into the backs of mice, mineralized bone tissues were formed 9 weeks following implantation. These hydrogels may find wide utility as an injectable delivery system for bone precursor cells as well as for other applications in tissue engineering.