Characterization of Gelatin Hydrogel Incorporating Growth Factor

In vivo degradation of gelatin hydrogels was evaluated in terms of the loss of radioactivity of implanted 125I-labeled gelatin hydrogels. Gelatin hydrogels were radioiodinated using 125I-Bolton-Hunter reagent. Briefly, 100 ^L of 125I-Bolton-Hunter reagent solution in anhydrous benzene was bubbled with dry nitrogen gas until benzene evaporation was completed. Then, 1 mL of 0.1 M sodium borate-buffered solution (pH 8.5) was added to the dried reagent to prepare an aqueous solution of 125I-Bolton-Hunter reagent. The aqueous solution was impregnated into freeze-dried disks of gelatin hydrogels at a volume of 20 ^L/disk. The resulting swollen hydrogels were kept at 4°C for 3 h to introduce 125I residues into the amino groups of gelatin. The radioiodinated gelatin hydrogels were placed in ddH2O, which was exchanged periodically at 4°C for 4 d to exclude noncoupled, free 125I-labeled reagent from 125I-labeled gelatin hydrogels. When measured periodically, the radioactivity of the ddH2O returned to a background level after 3 d of rinsing. The resulting swollen hydrogels were freeze-dried. In vivo degradation of gelatin hydrogels was evaluated in terms of the loss of radioactivity of implanted 125I-labeled gelatin hydrogels. Various types of 125I-labeled gelatin hydrogels were implanted into the back subcutis of ddY mice (three mice per group, 6 to 7 wk old). At 1, 3, 5, 7, 10, 14, and 21 d after hydrogel implantation, the radioactivities of explanted hydrogels were measured on a gamma counter. Next, the mouse back skin around the hydrogel site was cut into a 3 x 5 cm strip, and the corresponding facia site was thoroughly wiped off with filter paper and measured to evaluate the remaining radioactivity of tissue around the implanted hydrogel. The ratio of total radioactivity measured to the radioactivity of the initially implanted hydrogel was expressed as the percentage of remaining activity for hydrogel degradation.

An aqueous solution of 125I-labeled bFGF was sorbed into freeze-dried gelatin hydrogel disks to prepare gelatin hydrogel incorporating 125I-labeled bFGF. 125I-labeled bFGF was prepared according to the chloramines T method reported previously (18). Various types of gelatin hydrogels incorporating 125I-labeled bFGF were implanted into the backs of mice. An aqueous solution of 125I-labeled bFGF was subcutaneously injected into the backs of mice. At different time intervals, areas of mouse skin containing the implanted hydrogels or directly injected 125I-labeled bFGF were thoroughly wiped off with filter paper in a way similar to that just described. The radioactivities of the residual gelatin hydrogels and the skin strip plus filter paper were measured on a gamma counter, and their radioactivity ratios to the bFGF initially used were expressed as the percentage of remaining activity for in vivo bFGF release.

Recently, much research has been devoted to tissue regeneration through combinations of growth factors with various carrier materials (Table 2).

Table 2

Experimental Trials for Tissue Regeneration by Combination of Growth Factor With Carrier3

Table 2

Experimental Trials for Tissue Regeneration by Combination of Growth Factor With Carrier3

Growth factor

Carrier

Animal

Tissue regenerated

BMP

PLA

Dog

Long bone

Collagen sponge

Rat

Long bone

Dog, monkey

Periodontal ligament

and cementum

ß-TCP

Rabbit

Long bone

Porous HA

Rabbit

Skull bone

rhBMP-2

Porous PLA

Dog

Spinal bone

Rat

Skull bone

PLA microsphere

Rabbit

Skull bone

Collagen sponge

Dog

Periodontium

Gelatin

Rabbit

Skull bone

PLA-coating gelatin sponge

Dog, monkey

Long bone, jaw bone,

skull bone

Porous HA

Monkey

Skull bone

PLA-PEG copolymer

Rat

Long bone

rhBMP-7

Collagen

Dog

Spinal bone

Dog

Long bone

EGF

Agarose

Hamster

Angiogenesis

PVA

Rat

Dermis

aFGF

PVA

Mouse

Angiogenesis

Alginate

Mouse

Angiogenesis

bFGF

Alginate

Mouse

Angiogenesis

Agarose/heparin

Mouse, pig

Angiogenesis

Amylopectin

Mouse

Angiogenesis

Gelatin

Mouse

Angiogenesis, dermis

adipogenesis

Rabbit, monkey

Skull bone

Dog

Nerve

Fibrin gel

Mouse

Angiogenesis

Collagen minipellet

Rabbit

Long bone

Collagen

Mouse

Cartilage

Poly(ethylene-co-vinyl acetate)

Rat

Nerve

NGF

Collagen minipellet

Rabbit

Nerve

PLGA

Rat

Nerve

TGF-ß1

PEG

Rat

Dermis

Gelatin

Rabbit

Skull bone

Plaster of Paris, PLGA

Rat

Skull bone

TCP

Dog

Long bone

Porous HA

Dog

Long bone

Collagen

Baboon

Skull bone

Mouse

Dermis

PDGF-BB

Porous HA

Rabbit

Long bone

Collagen

Rat

Dermis

Chitosan

Rat

Periodontal bone

VEGF

Collagen

Mouse

Angiogenesis

Alginate

Mouse

Angiogenesis

HGF

Gelatin

Mouse

Angiogenesis

IGF-1

PLGA-PEG

Rat

Adipogenesis

IGF-1/bFGF

PLGA-PEG

Rat

Adipogenesis

PDGF/IGF-1

Titanium implant

Dog

Jaw bone

aaFGF, acid fibroblast growth factor; bFGF, basic fibroblast growth factor; BMP, bone morphoge-netic protein; EGF, epidermal growth factor; HA, hydroxyapatite; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; NGF, nerve growth factor; PDGF-BB, platelet-derived growth fac-tor-BB; PEG, poly(ethylene glycol); PLA, polylactide; PLGA, glycolide-lactide copolymer; PVA, poly(vinyl alcohol); rhBMP, recombinant human bone morphogenetic protein; TCP, tricalcium phosphate; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

All results claim the necessity of combining the growth factors with carriers to induce in vivo tissue regeneration. In addition to proteinaceous growth factor, the gene encoding growth factor has recently been applied to promote tissue regeneration (19). If the corresponding gene is transfected into the cells existing in the site of regeneration, it is highly possible that the cells can secrete the growth factor for a certain time period, resulting in promoted tissue regeneration. The angiogenetic therapy of ischemic diseases (20) and bone tissue regeneration (21) have been attempted by using the corresponding growth factor genes.

4. Notes

1. Characteristics of gelatin hydrogel for controlled release of growth factor. One of the largest problems in protein release technology is the loss of biological activity of proteins released from protein-carrier formulations. It has been demonstrated that this loss of activity results mainly from denaturation and deactiva-tion of proteins during preparation of carrier formulations. Therefore, a method to prepare protein release carriers with inert biomaterials should be exploited to minimize protein denaturation. From this viewpoint, a polymer hydrogel may be a preferable candidate for use as a protein release carrier because of its biocompatibility and its high inertness toward protein drugs.

We have created a release system for growth factors that mimics the native mode of growth factor delivery in the living body. Figure 2 shows a conceptual illustration for the controlled release of growth factor from a biodegradable polymer hydrogel based on physicochemical interaction forces between the growth factor and polymer molecules. For example, a hydrogel is prepared from a biodegradable polymer with negative charges. The growth factor with a positively charged site is electrostatically attracted to the polymer chain and thereby is physically immobilized in the hydrogel carrier. If an environmental change, such as increased ionic strength, occurs, the immobilized growth factor is released from the factor-carrier formulation. Even if such an environmental change does not take place, degradation of the carrier itself also leads to growth factor release. Because the latter is more likely to happen in vivo than the former, it is preferred that the release carrier be prepared from biodegradable polymers. This complex-ation protects the growth factor from denaturation and enzymatic degradation in vivo.

As the material for growth factor release, we have selected gelatin because it has the desired physicochemical properties and has been extensively used for industrial, pharmaceutical, and medical purposes. The biosafety of gelatin has been proven through its long clinical uses. Another unique advantage is the electrical nature of gelatin, which can be changed by the processing method. For example, an alkaline process of collagen results in hydrolysis of amide groups of the asparagine and glutamine residues, having a high density of carboxyl groups, which makes the gelatin negatively charged. The pi of gelatin is about 5.0.

Fig. 2. Conceptual illustration of growth factor release from a biodegradable hydrogel based on physical interaction forces.

By contrast, the nature of a gelatin is much different for an acidic process. A positively charged gelatin of "basic" type is prepared and the p/ is about 9.0. If a growth factor to be released has the positively charged site in the molecule that interacts with acidic polysaccharides present in the ECM, the negatively charged gelatin of "acidic" type is preferable as the carrier material. Considering the electrostatic interaction, the "basic" gelatin is preferable for release of molecules with the negative charged site. It was found that, as expected, bFGF, transforming growth factor-pi (TGF-pi), or platelet-derived growth factor was sorbed into the acidic gelatin hydrogel mainly owing to the electrostatic interaction (22).

Animal experiments revealed that the hydrogels prepared from the acidic gelatin were degraded in the body (23). The degradation period of hydrogels depends on their water content, which is a measure of crosslinking extent: the higher the water content of the hydrogels, the faster their in vivo degradation. The water content of hydrogels increased with increasing concentrations of chemical crosslinking agents and gelatin concentration used in the preparation of the hydrogels. The time profile of in vivo bFGF retention was in accordance with that of hydrogel degradation, irrespective of the hydrogel biodegradability. It seems reasonable to suppose that bFGF was released from the gelatin hydro-gel along with degraded gelatin fragments in the body as a result of hydrogel degradation. These findings strongly indicate that growth factor release is governed mainly by hydrogel degradation, as described in Fig. 2. As a result, in this release system, the release period is not influenced by the hydrogel's shape at all, but controllable only by changing the degradation rate of hydrogel (24). Note that gelatin hydrogels can be formed into different shapes of disks, tubes, sheets, granules, and microspheres (24,25).

2. Tissue regeneration by gelatin hydrogel incorporating bFGF. As described in the previous section, the gelatin hydrogel was found to be a superior carrier for the controlled release of growth factor. Hereafter, concrete experimental results on angiogenesis, bone regeneration, and adipogenesis are described as achieved by this release system alone or in combination with stem cells.

3. Angiogenesis. bFGF has been reported to have a variety of biological functions (26) and to be effective in enhancing wound healing through induction of angio-genesis and regeneration of bone, cartilage, and nerve tissue. Among its biological actions, the gelatin hydrogel was effective in enhancing the in vivo angiogenic effect of bFGF. When gelatin hydrogels incorporating bFGF were subcutane-ously implanted into a mouse's back, the angiogenic effect was observed around the implanted site, in marked contrast to the sites implanted with bFGF-free, empty gelatin hydrogels or injected with an aqueous solution of bFGF (22). No angiogenesis was induced by the injection of bFGF solution even when the dose was increased to 1 mg/site. This result must be owing to a rapid elimination of bFGF from the injection site (27). By contrast, the gelatin hydrogel incorporating bFGF induced significant angiogenesis even when the dose was as low as 30 ^g/site. The maintenance period of the hydrogel-induced angiogenic effect could be changed by prolonging the hydrogel's water content as the water content became lower (27). It is likely that the hydrogels with lower water contents were more slowly degraded and consequently released bFGF of biological activity in vivo less rapidly than those with higher water contents, leading to a prolonged angiogenic effect. A similar enhanced and prolonged angiogenic effect was also observed when using gelatin hydrogels incorporating bFGF of the microsphere type (24).

The technology to induce artificially in vivo angiogenesis is indispensable for tissue engineering. Two objectives of angiogenesis induction include the therapy of ischemic disease and advanced angiogenesis for cell transplantation. As an example of the former, the therapy of ischemic myocardium by gelatin hydrogels incorporating bFGF is introduced here. Myocardial infarction was induced by ligating the left anterior descending (LAD) coronary artery of dog heart. Gelatin microspheres incorporating bFGF were intramuscularly injected into both sides of the LAD 10 mm distal from the ligated site. As a control, an aqueous solution of bFGF at the same dose level was injected. Injection of the gelatin microspheres containing bFGF induced regeneration of collateral coronary arteries at the site of ligated LAD and increased the blood flow in the left circumflex coronary artery (LCX) (Fig. 3). More interesting, the injection of microspheres was also effective in recovering the motion of myocardium in the ischemic region. Neither of these therapeutic effects were observed for the injection of bFGF solution at the same dose level (1).

There is no doubt that a sufficient supply of nutrients and oxygen to the cells transplanted in the body is indispensable for cell survival and the maintenance of biological functions. Without sufficient supply, cells preseeded in a scaffold for tissue regeneration would hardly survive following implantation of the scaffold

Fig. 3. Left coronary angiograms of ischemic dog heart 1 wk after intramyocardial injection of (A) bFGF solution and (B) gelatin microspheres incorporating bFGF. bFGF was bilaterally injected at the distal side of the LAD ligated portion (indicated by Mark II) at a dose of 100 |g/heart. The hydrogel water content was 95.0 wt%.

Fig. 3. Left coronary angiograms of ischemic dog heart 1 wk after intramyocardial injection of (A) bFGF solution and (B) gelatin microspheres incorporating bFGF. bFGF was bilaterally injected at the distal side of the LAD ligated portion (indicated by Mark II) at a dose of 100 |g/heart. The hydrogel water content was 95.0 wt%.

into the body. Such a situation is caused by allo- or xenogeneic cells transplanted into the body for organ substitution. For successful cell transplantation, the nutrient and oxygen supply is the dominant challenge relative to immunoisolation. For both of the supplies, it is promising to induce angiogenesis throughout the transplanted site of cells by using angiogenic growth factors. In a recent study, pancreatic islets were encapsulated by a hydrogel bag effective for immunoisolation and implanted into the sc tissue of streptozotocin-induced diabetic mice. Advanced angiogenesis at the site of cell transplantation induced by gelatin microspheres containing bFGF enabled the encapsulated islets to improve the survival rate, resulting in a prolonged maintenance period of normal glucose level in the blood (Fig. 4) (28). This finding demonstrates that in vivo angiogen-esis induced by the gelatin microspheres containing bFGF could be achieved in the subcutis of even diabetic mice, which have an inferior injury-repairing capability relative to healthy mice. It is of prime importance to induce tissue regeneration even in the bodies of patients who have diseases or are elderly. Little tissue engineering research has been performed on aged animals. This area of study will undoubtedly become important when considering clinical applications for tissue engineering. This angiogenic effect for the prolonged cell survival was observed for transplantation of hepatocytes (29) and cardiomyocytes (30).

4. Bone regeneration. Gelatin hydrogels incorporating bFGF were found to have a promising potential for bone repair (31,32). For example, when implanted into a monkey skull defect, the gelatin hydrogel incorporating bFGF promoted bone

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0 7 IT tt 31 49 39 TO W flJ 101 \\l Time- ofPoitTnuijplQJil Jdjys]

Fig. 4. Time course of glucose level in blood of diabetic mice after sc xenograft of rat pancreas islets encapsulated by an agarose/PSSa hydrogel membrane into vascu-larized site induced by advance injection of gelatin microspheres containing bFGF (small arrow, transplantation). Two of 10 recipients became hyperglycemic again when the grafts were respectively retrieved at d 31 (O, *, and □) and d 63 (O, x, A, +, ♦, and ■) (large arrows). This strongly indicates that the encapsulated islets functioned normally in the subcutis of diabetic mice. The average normoglycemic period was 68.4 ± 25.6 d.

regeneration at the defect and closed the defect by 21 wk after implantation. By contrast, both the use of bFGF-free gelatin hydrogels and the use of a similar dose of bFGF in the solution resulted in a total lack of bone regeneration, and a remarkable ingrowth of soft connective tissues at the bone defect. Measurement of bone mineral density (BMD) at the skull defect revealed that gelatin hydrogels containing bFGF enhanced the BMD to a significantly higher extent than did free bFGF, irrespective of the hydrogel water content. The BMD resulting from the bFGF-free gelatin hydrogel implant was similar to that of the untreated group, indicating that the presence of hydrogel did not impair bone healing at the defect. In a histological study, hydrogel implantation increased the number of osteo-blasts residing near the edge of the bone defect and retained it at a significantly high level over the time range studied.

It is known that both TGF-P1 and bone morphogenetic protein (BMP) also promote bone regeneration (33-36). We have succeeded in repairing bone and skull defects of rabbits and monkeys by the controlled release of TGF-P1 from gelatin hydrogels, in marked contrast to the use of free TGF-P1 even at higher doses (34). However, the degree of repair depended on the water content of hydrogels, which could be reduced or increased. It is possible that too rapid degradation of the hydrogel causes a short period of bFGF release, resulting in no induction of bone regeneration. Conversely, a long-term residue of hydrogels owing to slow degradation would physically hinder bone regeneration. As a result, it is likely that the hydrogel with an optimal biodegradability induced complete bone regeneration at the skull defect (34). As with previously described studies, hydrogels function as carriers of growth factors as well as barriers to prevent the ingrowth fibrous tissues into bone defects. Balance of the time course between the two hydrogel functions would result in better bone repairing. We have recently succeeded in the controlled release of BMP-2 by hydrogels of a gelatin type. This controlled-release system enabled BMP-2 to induce formation of bone tissue ectopically or orthotopically at doses lower than used for the application of free BMP-2 in solution.

There are some cases in which a combination of the controlled release of growth factor and stem cells is effective in achieving bone repair. In one trial, we utilized cells with osteogeneic potentials and combined them with the growth factor release system. MSCs were isolated from the bone marrow of a rabbit fibula. We demonstrated that application of a combination of MSCs and gelatin microspheres containing TGF-P allowed completely repaired defects in rabbit skulls by newly formed bone tissue, in marked contrast to that of either material used alone (37). In this case, however, the TGF-P release system when used alone was not effective because the dose was too low.

5. Adipogenesis. When gelatin microspheres incorporating bFGF were mixed with a basement membrane extract (Matrigel) and subcutaneously implanted into a mouse's back, de novo formation of adipose tissue was observed at the implanted site (38). Recently, we also succeeded in inducing de novo adipogenesis by combining preadipocytes isolated from fat tissues, gelatin microspheres incorporating bFGF, and a collagen sponge (Fig. 5). When the preadipocytes and the microspheres were placed into the collagen sponge and implanted into the back subcutis of a mouse, de novo formation of adipose tissue was observed at the implanted sponge site. Combination of all three materials was needed to induce this adipogenesis (39). These results experimentally justify the strategy of in vivo tissue engineering asserting that tissue regeneration can be achieved by creating a suitable environment in the body site to be regenerated.

6. Conclusion. For regeneration of body tissues, a variety of growth factors act on cells by forming a complex network while the action timing, action site, and concentration of growth factors are delicately regulated in the body. It is likely that the mechanisms of tissue regeneration in living systems will be clarified with rapidly advancing progress in cell biology, molecular biology, and embryology. Even so, it will be impossible to imitate living systems by solely making use of the scientific knowledge and technologies currently available. However, clarification of living mechanisms will help researchers to understand which growth factor is key to induce the regeneration of a target tissue. If such a key growth factor is supplied to the necessary site at a suitable time period and concentration, I believe that the living body will be stimulated toward the process of natural tissue regeneration. Once the right direction toward tissue regeneration is

Fig. 5. De novo formation of adipose tissue in mouse subcutis 6 wk after implantation of a collagen sponge containing a mixture of preadipocytes and gelatin microspheres incorporating bFGF: (A) collagen sponge containing mixture of preadipocytes and gelatin microspheres incorporating bFGF; (B) collagen sponge containing mixture of preadipocytes and free bFGF; (C) collagen sponge containing preadipocytes; (D) mixture of preadipocytes and gelatin microspheres incorporating bFGF; (E) collagen sponge containing gelatin microspheres incorporating bFGF. (Magnification: x100; Sudan III staining). The gelatin microspheres incorporating bFGF were completely degraded to disappear from the injected site. The bFGF dose was 10 |g/site and the hydrogel water content was 95.0 wt%. Bar = 300 |im. CS, collagen sponge; AT, adipose tissue newly formed; C, capillary newly formed.

Fig. 5. De novo formation of adipose tissue in mouse subcutis 6 wk after implantation of a collagen sponge containing a mixture of preadipocytes and gelatin microspheres incorporating bFGF: (A) collagen sponge containing mixture of preadipocytes and gelatin microspheres incorporating bFGF; (B) collagen sponge containing mixture of preadipocytes and free bFGF; (C) collagen sponge containing preadipocytes; (D) mixture of preadipocytes and gelatin microspheres incorporating bFGF; (E) collagen sponge containing gelatin microspheres incorporating bFGF. (Magnification: x100; Sudan III staining). The gelatin microspheres incorporating bFGF were completely degraded to disappear from the injected site. The bFGF dose was 10 |g/site and the hydrogel water content was 95.0 wt%. Bar = 300 |im. CS, collagen sponge; AT, adipose tissue newly formed; C, capillary newly formed.

taken, the intact system of the body will start to act and function, resulting in automatic achievement of tissue regeneration. There is no doubt that as long as growth factors are used, their controlled release will be an essential technology in the future. Recently, bFGF has been on the Japanese market as a therapeutic agent for skin ulcer and decubitus. I expect that this will be a cue to encourage clinical application of tissue regeneration based on growth factor DDS technology.

If tissue engineering matures to be the third choice of therapeutic medicine relative to reconstructive surgery and organ transplantation, it will give patients many therapeutic choices and privileges. To this end, substantial collaborative efforts among researchers in materials, pharmaceutical, biological, and medical sciences, and clinical medicine are needed to reach academic and technical maturity in tissue engineering. Because tissue engineering is still in its infancy, it will take much more time before its full potential is realized. Without the cell scaffold and DDS technologies to induce tissue regeneration, any developmental results of medicine, biology, and molecular biology regarding stem cells will never be realized in medical therapy for patients, which is the final goal of "regenerative" medicine. Tissue engineering is one of the indispensable tools to make regenerative medicine clinically available. Little DDS research aiming at tissue regeneration as well as organ substitution has been conducted. I am confident that the majority of the readers of this chapter will get a better understanding of the magnified significance of biomaterials as well as DDSs in the future progress of tissue engineering. It is hoped that this article will increase readers' interest in this research field.

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