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J Dent Res 82(11): 903-908, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Regulating Bone Formation via Controlled Scaffold Degradation

E. Alsberg1, H.J. Kong2, Y. Hirano3, M.K. Smith2, A. Albeiruti3, and D.J. Mooney1,2,3,*

1 Departments of Biomedical Engineering,
2 Chemical Engineering, and
3 Biologic and Materials Sciences, Room 5213 Dental School, 1011 North University, University of Michigan, Ann Arbor, MI 48109-1078, USA;

* corresponding author, mooneyd{at}umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

KEY WORDS: alginate • biomaterials • tissue engineering • irradiation • osteoblasts


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scaffolding materials for tissue engineering applications are typically designed to provide a space for transplanted or infiltrating cells to attach, proliferate, and produce new extracellular matrix (Putnam and Mooney, 1996). In bone tissue engineering in particular, the deposition of a matrix, and its subsequent mineralization, is required for the development of the tissue, regardless of whether conductive, inductive, or cell transplantation approaches are utilized to form the new bone (Alsberg et al., 2001b). It is widely assumed that the materials used for tissue formation in these approaches should degrade over time in the body to provide new space for this matrix deposition (Nerem and Sambanis, 1995; Pollok and Vacanti, 1996), and to allow the tissue forming around each cell or cell cluster to coalesce into one interconnected tissue structure to increase the mechanical function of the tissue. In vitro studies demonstrate that an increased polymer biodegradation rate can affect cell proliferation/survival and extracellular matrix distribution (Bryant and Anseth, 2002). However, little direct evidence exists to support a relation between the degradation rate of the scaffolding and either the quantity or quality of an engineered tissue in vivo.

This study addressed the hypothesis that modifying the degradation rate of polymeric materials utilized to transplant bone-forming cells and to engineer bony tissues will directly control the extent of new bone formation. We tested this hypothesis by transplanting osteoblasts into mice using an alginate hydrogel system. Previous studies have demonstrated that alginate hydrogels are useful for the engineering of bony and cartilaginous tissues (Paige et al., 1995; Diduch et al., 2000). These materials are especially attractive for clinical application, since they can be used to transplant cells in a minimally invasive manner, and alginate has been safely used to deliver proteins and cells to patients (Bent et al., 2001; Bratthall et al., 2001). Further, cell adhesion peptides may be covalently coupled to alginate (Rowley et al., 1999), to control the mechanism of cell adhesion and phenotype in vitro (Alsberg et al., 2001a; Rowley and Mooney, 2002) and bone and cartilage formation in vivo (Alsberg et al., 2001a, 2002).

The molecular weight and structure of alginate polymers provide a readily manipulated means for regulation of the degradation rate of hydrogels formed from these polysaccharides. Alginate is comprised of mannuronic (M) and guluronic (G) sugars organized into blocks of M-residues, blocks of G-residues, and blocks comprised of alternating residues, and will form space-filling hydrogels by ionic cross-linking with divalent cations such as calcium. Only the G-blocks participate in ionic cross-linking, and a minimum number of G-residues/block is necessary for cross-linking (Smidsrod and Skjak-Braek, 1990). Main chain scission of alginate at a neutral pH is minimal (Lansdown and Payne, 1994; Draget et al., 1997). Instead, these gels dissolve slowly upon losing divalent cations (Bouhadir et al., 2001b). We hypothesized that the molecular weight of alginate will regulate chelation of the cross-linking calcium and gel dissolution in vivo. In this study, alginate has been irradiated to control these features and test their role in bone tissue engineering.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In vivo Polymer Degradation
MVG sodium alginate powder (Pronova Biopolymers, Oslo, Norway) with an apparent viscosity of 241 mPa•s and an average molecular weight of 259 kDa was lyophilized until dry at pH 7, and subjected to gamma-irradiation (0-20 Mrad) (Phoenix Lab, University of Michigan, Ann Arbor, USA). We used gel permeation chromatography (GPC) to measure the average molecular weight of the alginate chains before and after irradiation as previously described (Bouhadir et al., 2001a). The alginates were subsequently reconstituted in {alpha}-MEM and 100 units/mL PS (Gibco BRL, Gaithersburg, MD, USA), yielding 2% and 3% w/w solutions, and were sterile-filtered through a 0.22-µm syringe filter. The number of G-residues/G-block and the G-content were determined as previously described (Kong et al., 2002).

Gel disks (6 mm diameter, 2 mm thick) were formed as previously described (Rowley et al., 1999) from (1) 0 Mrad 2% alginate, (2) 5 Mrad 2% alginate, and (3) 8 Mrad 3% alginate, and were implanted subcutaneously into the backs of 4- to 5-week-old anesthetized male CB-17 SCID mice (Taconic Farms, Inc., Germantown, NY, USA). Disks were harvested and weighed at 2, 4, and 12 wks. The elastic moduli were measured (Kong et al., 2002), and samples that could not be positively identified were assumed to have negligible mass and negligible elastic moduli. Statistical analysis of the mass and elastic moduli data was performed by unpaired one-tailed Student’s t tests unless the difference between standard deviations was determined to be significant, in which case the one-tailed alternate Welsh t test was performed. Statistical significance was defined by P < 0.05.

Implantation of Osteoblasts into Peptide Modified Alginates
G4RGDSP peptides (Commonwealth Biotechnologies, Inc., Richmond, VA, USA) were covalently coupled to the alginates (Rowley et al., 1999) to achieve a peptide density of 1.5 x 105 nmol/L when alginates were reconstituted to a 2% w/w solution in {alpha}-MEM. This peptide type and density significantly increase the amount of in vivo bone formed compared with unmodified alginate (Alsberg et al., 2001a). Primary rat-derived calvarial osteoblasts (PRCO) were isolated (Pockwinse et al., 1992) from 1- to 3-day-old newborn Lewis rats (Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) and mixed at a concentration of 24.5 x 106 cells/mL with (1) non-irradiated alginate-G4RGDSP and (2) 8-Mrad-treated alginate-G4RGDSP. Cell-alginate constructs were cross-linked with calcium sulfate, and 200 µL per implant was injected into the backs of 4- to 5-week-old anesthetized male CB-17 SCID mice as previously described (Alsberg et al., 2001a).

Implants were harvested at 6 (N = 5), 13 (N = 5), and 21 (N = 5) wks, weighed, imaged on a DEXA scanner for determination of bone mineral density (BMD) and bone mineral content (BMC), and subjected to constant strain rate compression tests at 21 wks (N = 4). Samples were assayed for calcium (Calcium Kit, Sigma, St. Louis, MO, USA), phosphate (Heinonen and Lahti, 1981), and DNA content (Schneider, 1957). The 6- and 13-week specimens were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H/E), and the 21-week specimens were plastic-embedded (Pathology Associates, West Chester, OH, USA), sectioned, and stained with Goldner’s Trichrome. Histomorphometric analysis was performed for quantitative determination of bone fraction as previously described (Alsberg et al., 2002). Specimens at 21 wks were imaged at a resolution of 9 µm by means of a three-dimensional microcomputed tomography (µCT) system and reconstructed at a resolution of 18 µm for qualitative examination of the microarchitecture of the engineered bone tissue. Statistical comparison of bone formation in the alginate delivery vehicles was performed by paired one-tailed Student’s t tests, and statistical significance was defined by P < 0.05. A protocol based on NIH guidelines for the care and use of laboratory animals (NIH Publication #85-23 Rev 1985) was reviewed and approved by an institutional review board and was observed in all procedures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Polymer Degradation
GPC analysis demonstrated that irradiation decreased the molecular weight of the alginate in a dose-dependent manner (Fig. 1AGo), and irradiation at 8 Mrad or below maintained a constant M:G ratio and constant G-block size. The number of G-residues in one G-block for non-irradiated and 8-Mrad-irradiated alginate was 30 and 29, respectively. The fraction of G-residues in each alginate molecule was 0.78 for both non-irradiated and 8-Mrad-irradiated alginate. Analysis of these data indicates that chain scission is occurring mainly at the bonds formed between M- and G-residues at this dose, and the preservation of both overall G-content and G-block length maintained the gel-forming ability of these polymers. In contrast, alginate irradiated at higher doses demonstrated a decrease in the G-block length, along with the decreased molecular weight, and formed extremely soft, weak gels.



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  		Figure 1. Gamma-irradiation decreases the average molecular weight of alginate polymer chains and increases its biodegradation rate in vivo. (A) Average molecular weight of alginate as a function of radiation dose. Experimental values are reported as mean (N = 3) ± SD. Gross appearance of explanted gels formed from (B) 0 Mrad 2% alginate and (C) 8 Mrad 3% alginate after 12 wks’ subcutaneous implantation in the backs of SCID mice. [Size bar in photomicrographs (B) and (C) represents 6 mm.] Photomicrographs of H/E-stained histological sections of (D) non-irradiated and (E) 8-Mrad-irradiated hydrogels at 12 wks. [Size bar in photomicrographs (D) and (E) represents 200 µm. A = residual alginate, F = fibrous tissue.] (F) Change in implant mass over time. [Experimental values are reported as mean (N = 4-5) ± SD. (*) indicates a statistically significant (P < 0.05) difference in implant mass between irradiated alginate and non-irradiated alginate control at a specific time point. ({blacksquare}) 0 Mrad 2% alginate, (•) 5 Mrad 2% alginate, ({blacktriangleup}) 8 Mrad 3% alginate.]

 
The degradation of gels formed from these polymers was then tested by implantation in mice. Experimental conditions were chosen to meet two criteria: (1) the molecular weight of the irradiated alginate was at or below 50 kDa, to allow for ultimate clearance by the kidneys (Al-Shamkhani and Duncan, 1995); and (2) the hydrogels could be formed with sufficient mechanical integrity to allow for implantation without damage to the constructs. Alginate exposed to 5 or 8 Mrad gamma-irradiation met these conditions and was tested along with non-irradiated alginate as a control. All non-irradiated alginates (N = 5 at each time point) kept their shape and were positively identified (Fig. 1BGo). In contrast, the 5-Mrad-irradiated samples did not keep their initial shape, shrunk, and demonstrated fibrous tissue ingrowth. The 8-Mrad-irradiated samples were extremely hard to identify at all harvest time points, did not keep their shape, shrunk, fragmented, and demonstrated extensive fibrous tissue ingrowth (Fig. 1CGo). Histological examination revealed large islands of residual alginate with little fibrous tissue ingrowth in the non-irradiated specimens (Fig. 1DGo), but much less residual alginate in the 8-Mrad-irradiated specimens (Fig. 1EGo). Similarly, little decrease in mass (Fig. 1FGo) and no decrease in elastic moduli were observed in the non-irradiated implants. In contrast, significant mass and moduli decreases in the irradiated alginate implants indicated biodegradation of these materials. The implant mass at 12 wks increased, likely due to fibrous tissue ingrowth.

Bone Formation
The ability of alginate hydrogels of various degradation rates to allow bony tissue to form from primary rat calvarial osteoblasts (PRCO) was next tested with the use of non-irradiated alginate and alginate irradiated with 8 Mrad. More rapidly degrading gels allowed for improved bone formation, since these samples grossly demonstrated the presence of a mineralized, vascularized tissue which was stiffer than the non-irradiated samples. Histological examination of the non-irradiated alginate implants revealed strips of bone tissue that grew in size over time, but large islands of residual alginate remained at all times (Figs. 2A, 2C, 2EGo). In contrast, the irradiated specimens were comprised of many small islands of residual alginate surrounded by new tissue (Fig. 2BGo), and the residual alginate islands decreased in size and the bony trabeculae grew thicker and more interconnected over time (Figs. 2D, 2FGo). The non-irradiated alginate condition maintained its mass throughout the duration of the study (Fig. 3AGo). In contrast, the irradiated specimens decreased their mass by almost 50% after 6 wks and then gradually increased in mass to a value statistically similar to that of the control at 21 wks. The initial decrease may be attributable to alginate degradation, and the subsequent increase to new bone formation. DEXA imaging revealed that the BMC significantly increased over time in the irradiated alginate group and increased modestly in the non-irradiated control (Fig. 3BGo). BMD significantly increased over time in both groups, but was significantly greater in the irradiated group than in the non-irradiated control at 21 wks (Fig. 3CGo). Chemical assays demonstrated that phosphate and calcium content significantly increased over time in both experimental groups, but there was significantly more phosphate and calcium content in the irradiated alginate condition compared with the non-irradiated control at 13 wks (data not shown). Hydroxyapatite (Ca10(PO4)6(OH)2), the mineralized portion of native bone, has a calcium-to-phosphate molar ratio of 1.67, and a similar ratio was found in both experimental groups at 13 and 21 wks. Histomorphometric analysis confirmed that the increases in mineral content were a direct result of increased bone tissue formation, and at all time points, significantly more bony tissue was formed in the irradiated alginate group compared with the non-irradiated control (Fig. 3DGo).



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  		Figure 2. Transplantation of osteoblasts in alginate with increased degradation rate resulted in qualitatively improved bone formation. Photomicrographs of (A-D) H/E and (E-F) Goldner’s Trichrome-stained histological sections of tissues formed from primary rat calvarial osteoblasts transplanted in (A,C,E) 0 Mrad 2% alginate-G4RGDSP and (B,D,F) 8 Mrad 2% alginate-G4RGDSP after 6, 13, and 21 wks’ subcutaneous implantation. [Size bar in photomicrographs represents 200 µm. A = residual alginate, B = bone tissue.]

 


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  		Figure 3. Transplantation of osteoblasts in alginate with increased degradation rate resulted in quantitatively improved bone formation. Change in tissue-engineered (A) bone mass, (B) BMC, and (C) BMD over time. (D) Histomorphometric quantification of implant bone fraction over time. [Experimental values are reported as mean (N = 4-5) ± SD. (*) indicates a statistically significant (P < 0.05) difference between irradiated alginate and non-irradiated alginate control at a specific time point.]

 
The quality of the engineered bone was also assessed by µCT and mechanical testing. µCT images of these specimens indicate increased bony tissue, thicker bony trabeculae, and increased connectivity of the bone microarchitecture in the irradiated alginate condition, as compared with the non-degrading alginate condition (Fig. 4Go). Constant strain rate compression tests revealed significantly higher elastic moduli in the irradiated condition (1.94 ± 0.87 MPa) compared with the non-irradiated control (0.32 ± 0.22 MPa).



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  		Figure 4. Increased polymer degradation rate results in improved bone microarchitecture as demonstrated by µCT images. µCT image slices of (A) 0 Mrad 2% alginate-G4RGDSP + PRCO and (B) 8 Mrad 2% alginate-G4RGDSP + PRCO after 21 wks. [Image reconstructions are at a resolution of 18 µm; size bars represent 3 mm.]

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study indicate that the degradation rate of vehicles used for transplanting bone-forming cells dramatically influences subsequent bone formation. Non-irradiated alginate degraded slowly, and a limited amount of bony tissue was formed. The irradiated alginate, however, degraded more quickly, and permitted more rapid development of a bony tissue that was structurally superior to that created with the use of more slowly degrading alginate. The moduli of the irradiated alginate constructs were only one order of magnitude lower than that found in normal trabecular bone (50-100 MPa) (Yaszemski et al., 1996). It is important to note, however, that biomaterials which biodegrade too rapidly may not be able to serve as a space-filling scaffold capable of supporting new tissue development.

Hydrogels composed of high-molecular-weight alginate exhibit limited biodegradation (Lansdown and Payne, 1994; Shapiro and Cohen, 1997), likely as a result of slow exchange of divalent cation cross-linkers with monovalent cations present in the environment surrounding the hydrogels. Several techniques have been reported to reduce the molecular weight of alginate, including radiation (Kume and Takehisa, 1983; Nagasawa et al., 2000), acid (Haug et al., 1966; Bouhadir et al., 2000), and enzyme treatment (Tsujino and Saito, 1961; Nakada and Sweeny, 1967; Shimokawa et al., 1996), and oxidation of alginate also regulates its biodegradation (Bouhadir et al., 2001b). Gamma-irradiation was utilized in these studies because it is a simple, readily controlled method that is unlikely to result in harmful by-products. The application of 8 Mrad or less of radiation treatment preferentially causes chain scission within MG-blocks (Kong et al., 2002), and the lengths of individual GG-blocks are unaltered. The radiation dose of 8 Mrad was chosen for cell transplantation experiments because it yielded a polymer molecular weight low enough to allow for ultimate renal clearance (Al-Shamkhani and Duncan, 1995), while still forming initially stable gels that rapidly degraded in vivo. The concentration of this alginate in the gels was varied between 2 and 3%, but this appeared to have little effect on the degradation rate (data not shown). A constant alginate concentration (2%) was utilized for the cell transplantation study, to be consistent with previous studies (Alsberg et al., 2001a, 2002).

This study illustrates that a polymer scaffold’s ability to degrade in concert with new tissue formation is a critical tissue engineering matrix parameter that allows for more rapid tissue formation and replacement with the desired tissue type. Control of both the degradation and adhesion characteristics of a polymer scaffold will be a powerful tool in regulating the regeneration processes of a broad range of tissues. The appropriate biomaterial degradation rate required for a specific tissue will likely be a function of the metabolic activity of the cell type(s) used for transplantation and the specific strategy for defect repair.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge funding from the NIDCR to DM (R01-DE13033) and EA (T32-DE07057), and thank Colleen Flanagan for assistance with µCT and Dr. Evan Keller for generously sharing his DEXA system.

Received March 27, 2003; Last revision July 30, 2003; Accepted August 4, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Al-Shamkhani A, Duncan R (1995). Radioiodination of alginate via covalently-bound tyrosinamide allows for monitoring of its fate in vivo. J Bioact Compat Polym 10:4–13.[Abstract]

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Alsberg E, Hill EE, Mooney DJ (2001b). Craniofacial tissue engineering. Crit Rev Oral Biol Med 12:64–75.[Abstract]

Alsberg E, Anderson KW, Albeiruti A, Rowley JA, Mooney DJ (2002). Engineering growing tissues. Proc Natl Acad Sci USA 99:12025–12030.[Abstract/Free Full Text]

Bent AE, Tutrone RT, McLennan MT, Lloyd LK, Kennelly MJ, Badlani G (2001). Treatment of intrinsic sphincter deficiency using autologous ear chondrocytes as a bulking agent. Neurourol Urodyn 20:157–165.[ISI][Medline]

Bouhadir KH, Kruger GM, Lee KY, Mooney DJ (2000). Sustained and controlled release of daunomycin from cross-linked poly(aldehyde guluronate) hydrogels. J Pharm Sci 89:910–919.[ISI][Medline]

Bouhadir KH, Alsberg E, Mooney DJ (2001a). Hydrogels for combination delivery of antineoplastic agents. Biomaterials 22:2625–2633.[ISI][Medline]

Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ (2001b). Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog 17:945–950.[Medline]

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