Claims:We Claim:

1. Hydrogel scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength.

2. Hydrogel scaffold as claimed in claim 1 wherein said cross-linked rigid biopolymer include alginate having ordered structure of self-assembled bubble based embedded voids of desired macroporosity, said voids formed of surfactant sustained bubble induced in said alginate.

3. Hydrogel scaffold as claimed in anyone of claims 1 or 2 comprising single phase blended or layered composite scaffold of at least two or more biopolymer including self-assembled multilayer voids with macroporosity of said voids in the order of at least 100 µm.

4. Hydrogel scaffold as claimed in anyone of preceding claims 1 to 3 comprising controlled macroporosity including series of self-aligned voids.

5. Hydrogel scaffold as claimed in anyone of preceding claims 1-4 comprising biopolymers selected from alginate, gelatin, chitosan or mixtures thereof.

6. Hydrogel scaffold as claimed in anyone of preceding claims 1-5 wherein said single phase blended scaffold comprises blend of two or more biopolymers including said self-aligned voids and layered scaffold includes sandwich type composite scaffold involving plurality of biopolymers layered atop one another including said self-aligned voids.

7. Hydrogel scaffold as claimed in anyone of preceding claims 1-6 including scaffold for drug delivery or tissue reconstruction with anyone or more induced characteristics selected from i) strength to sustain the loading on the scaffold until degradation occurs, ii) diffusion of biological entities in the scaffold at a suitable rate, iii) an induced desired macroporosity allowing cell colonization and vessel formation, to develop further the elastomeric qualities with tunable compressive moduli and strength, and to balance rate of resorption with the rate of tissue conduction.

8. Hydrogel scaffold as claimed in anyone of preceding claims 1-7 as blend scaffold comprising alginate and gelatin blend in the ratio of 1 to 4 : 1 to 4 preferably 3:2 with embedded voids, absorptivity of 1630 to 3400 % preferably about 3314.28% and mechanical strength of 0.003 to 0.05 preferably about 0.033 MPa as compared to void free alginate and gelatin scaffold having absorptivity at 2439.31%.

9. A process for the preparation of hydrogel scaffold comprising:
i) providing for biopolymer combination in blended and/or layered form;
ii) said biopolymer combination being obtained of atleast one polymer having tunable structure of induced multilayer voids of selectively desired anyone or more of macroporosity, elasticity and strength involving combination of ordered structure of self-assembled bubble based embedded voids of desired macroporosity obtained of surfactant sustained bubbles of aqueous suspension of said biopolymer prior to cross-linking and gelation.

10. A process for the preparation of hydrogel scaffold as claimed in claim 9 including blend or layered composite scaffold of cross-linked rigid biopolymer gel free of any chemical or thermal treatment comprising the steps of
a. providing an aqueous solution/suspension of biopolymer/s as homogeneous single phase solution free of any bubble;
b. adding surfactant to said homogeneous aqueous solution/suspension of biopolymer/s to suspend the mixture of biopolymers in a single phase;
c. introducing and embedding bubbles through a fluidic co-flow device to provide for self aligned bubble based polymeric film;
d. optionally, layering a biopolymer on said polymeric film to generate a sandwich type structure;
e. crosslinking and gelation followed by vacuum drying or optionally lyophilization to attain said hydrogel scaffold including blend or layered composite scaffold with desired macroporosity and intrinsic porosity.

11. A process for the preparation of hydrogel scaffold as claimed in claim 10 of a single biopolymer wherein said step (a) involves providing an aqueous solution of single biopolymer;
wherein said step (d) involves crosslinking with CaCl2 solution preferably a 4 wt% CaCl2 solution to obtain hydrogel scaffold of desired macroporosity and intrinsic porosity.

12. A process for the preparation of hydrogel scaffold as claimed in claim 10 as blended composite scaffold comprising the steps of
(a) providing a blended aqueous suspension of at least two or more biopolymers as single phase homogeneous solution free of any bubble preferably aqueous suspension of alginate and gelatin;
(b) adding surfactant preferably pluronic F-127 to said homogeneous aqueous suspension to retain it as a single phase suspension;
(c) introducing and embedding bubbles through said fluidic co-flow device providing for self aligned bubble based polymeric film preferably at liquid/ aqueous suspension flow rate of 5 mL/mim and a gas flow rate at 1 mL/min wherein said introduced bubbles could be sustained prior to crosslinking and gelation;
(d) crosslinking and gelling by crosslinking agent preferably 4 wt% CaCl2 solution followed by vacuum or freeze drying to obtain said blended composite scaffold therefrom of desired macroporosity.

13. A process for the preparation of hydrogel scaffold as claimed in claim 10 as layered composite scaffold comprising the steps of
(a) providing an aqueous solution of a biopolymer as a homogeneous solution free of any bubble preferably aqueous solution of alginate;
(b) adding surfactant preferably pluronic F-127 to said homogeneous aqueous solution of alginate;
(c) introducing and embedding bubbles through said fluidic co-flow device providing for self aligned bubble based polymeric alginate film preferably at aqueous solution flow rate of 5 mL/min and a gas flow rate at 1 mL/min wherein said introduced bubbles was sustained to provide for voids in the alginate gel that was preferably vacuum dried preferably for 2 hours to remove unbound moisture or the surface moisture from the polymeric gel film attained;
(d) optionally, layering a biopolymer preferably chitosan on both the sides of said polymeric alginate gel film prior to the development of gel structure of chitosan to generate a sandwich type composite structure;
(e) adding formaldehyde solution to chitosan solution for crosslinking and gelation followed by vacuum drying and lyophilization to attain said hydrogel scaffold as layered composite scaffold with desired macroporosity and intrinsic porosity.

14. A process for the preparation of hydrogel scaffold as claimed in claim 13
wherein in said step (d) preferably about 2 wt% chitosan solution in 0.2 N Acetic acid solution was layered on 2 wt% alginate gel with voids;
wherein in step (e) the composite structure film was vacuum dried at 40°C for about1 hour and was thereafter dried in a freeze drier for about 17 hours

15. A process for the preparation of hydrogel scaffold as claimed in anyone of claims 10-14 wherein said step (e) involves lyophilization preferably in a freeze drier conducted in three phases wherein in the first phase, the temperature of the scaffold was reduced to - 30°C to freeze the moisture present in the scaffolds without disturbing the gel strands, wherein in the second phase (sublimation) a vacuum of about 0.09 mbar (absolute) was imposed on the scaffold and the temperature increased to 0°C with the increase of temperature from -30°C following a ramp of 10°C/ 60 min, and a soak at -20°C, -10°C, and 0°C for 45 minutes, and wherein in the third phase (secondary drying) the temperature was increased to upto about 40°C through a similar ramp and soak sequence by maintain the vacuum all along favouring augmentation of intrinsic porosity of the gel matrix based hydrogel scaffold by retention of the pore structure during lyophilization.

Dated this the 21st day of January, 2016 Anjan Sen
Of Anjan Sen and Associates
(Applicants Agent)

, Description:FIELD OF THE INVENTION

The present invention provides for a hydrogel scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength and methods of preparation of the same. Particularly, the methods implemented in the present invention for the preparation of scaffold are selected from single phase blending of any one or more biopolymers including (i) blending of gelatin and alginate, and (ii) coating chitosan over alginate wherein a fluidic device was utilized to introduce self-aligned series of bubbles that left microvoids in the final structure said voids formed of surfactant sustained bubble induced in said biopolymer, which scaffold is advantageously suitable for end use and application such as delivering biological agents to the host tissue, providing a three-dimensional support for matrix formation.

BACKGROUND ART

Various methods of making porous gel scaffolds were reported in prior art [Chung, H.J., and Park, T.G.”Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering”, Advanced Drug Delivery Reviews 59(2007) 249-262; Turng, L.S., Kramschuster, A.J., “Method of fabricating a tissue engineering scaffold”, US Patent No. US 7,998,380 B2 dated Aug 16, 2011]. Freeze drying method for making of alginate scaffold has been patented (Zhang, M., Li, Z., “Porous structures, and methods of use”, US Patent No. US7736669 B2, Published on Jun 15, 2010). Use of microfluidic devices to generate gel scaffold is a recent development. Co-flow device in line with Utada, A.S. et al., “Monodisperse double emulsions generated from a microcapillary device”, Science 308 (2005) 537-541 has been used to generate bubbles in the alginate solution by Chung et at. in Chung,K., Mishra, N.C, Wang, C., Lin, F., and Lin, K.,” Fabricating Scaffolds by microfluidics”, BioMicrofluidics 3, 022403(2009).
A patent on different flow focusing methods, as apparatus of fluid dispersion in general has been granted to a large group of scientist [Stone, H.A., Anna, S.L., Bontoux, N., Link, D.R., Weitz, D.A., Gitlin, I., Kumacheva, E., Garstecki, P., Diluzio, R., Whitesides, M., “Method and apparatus for fluid dispersion. Patent No. US8,337,778 B2 dated Dec 25, 2012; Lin, K.H., Hong, W.J., “Method and device of fabricating three dimensional scaffolds”, US Patent No. US20110285047 A1, published on Nov 24, 2011]. A co-flow device, named as the “Orifice in Throat” has also been used to generate bubbles in the alginate solution by Ganguly et. al. [Bal, D.K., Patra, S., Ganguly, S. “Drying characteristics and evolution of the pore space in alginate scaffold with embedded sub-millimeter voids”, Journal of Sol-Gel Science Technology (2013) 68:254–260]. The alginate gel has been extensively studied for applications in controlled release [Kuo, C. K., and P. X. Ma. lonically crosslinked alginate hydrogels as scaffolds for tissue engineering: 1. Structure, gelation rate and mechanical properties. Biomaterials 22:511–521, 2001; Draget KI, Ostgaard K, Smidsrod O. Homogeneous Alginate Gels: A Technical Approach. Carbohyd Polym. 1991; 14: 159-178; Sheridan MH, Shea LD, Peters MC, Mooney DJ. Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J. Controlled Release 2000; 64: 91-102; Dohnal J, Stepanek F. Inkjet fabrication and characterization of calcium alginate microcapsules, Powder Technology 2010; 200:254–259; Li RH, Altreuter DH, Gentile FT. Transport characterization of hydrogel matrices for cell encapsulation. Biotechnol Bioeng.1996; 50(4): 365-73; Bal, D.K., Patra, S., Ganguly, S. “Use of orifice-in-throat device to make alginate scaffolds with embedded voids of sub-millimeter tunable dimensions”, Microsystem Technologies 20 (2014) 1359-1364; Ganguly, S., Bal, D.K., Patra, S., “Use of a co-flow device to make a gel matrix with embedded voids of sub-millimeter tunable dimensions”, Indian Patent filed No. 466/KOL/2013; Patra,S., Bal, D.K., Ganguly, S. “Bubble formation in complex fluids using an orifice in throat arrangement”, Experimental Thermal and Fluid Science 64 (2015) 62-69; Patra, S., Ganguly, S. “Bubble pinch-off in a cross-flowing biopolymer stream”, Microfluidics and Nanofluidics (2015) DOI 10.1007/s10404-015-1601-5.

The biomimicking of certain structures and properties of articular cartilage and Extracellular Matrix can be synchronously achieved by using layered scaffolds techniques [Zhu,Y, Wan, Y., Zhang, J., Yin, D., Chenge,W. “Manufacture of layered collagen/chitosan-polycaprolactone scaffolds with biomimetic microarchitecture” Colloids and Surfaces B: Biointerfaces 113 (2014) 352–360]. Many researchers (Mandal, B. B.; Mann, J. K.; Kundu, S. C.Silk fibroin/gelatin multilayered films as a model system for controlled drug release Eur. J. Pharm. Sci. 2009, 37, 160– 171; Schauer , C. L., Cathell, M.D., Mcilwee, H.A., “Multilayer films”, US Patent No. US20100062232 A1, Published on Jun 26, 2008; Harley B.A., Lynn A.K., Wissner-Gross Z., Bonfield W., Yannas I.V., Gibson L.J. “Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces” Journal of Biomedical Material Research Part A. 92(3) (2010) 1078-93) have studied the multilayer hydrogel films. Few patents were filed on multilayer scaffold (Sahoo, S. K., Chandana, M. “Process for Preparing Curcumin Encapsulated Chitosan Alginate Sponge Useful for Wound Healing”, Patent No. US20130171215 A1, Published on Jul 4, 2013; Lee, K. Y., Park, H. H., “Ionically cross-linkable alginate-grafted hyaluronate compound”, Patent No. US20150064143 A1, Published on Mar 5, 2015; Li, F., Rong T., Haibo S., Fang, X., “Alginate-gelatin-carboxymethylcellulose sodium blend membrane, and preparation and application thereof”, Patent No. CN102850598 B, Published on Feb 4, 2015; Yang., Duo, Z., Zhiqing, T., Xinyuan, S., “Oxidized sodium alginate/gelatin degradable hydrogel and preparation method thereof”, Patent No. CN102417734 B, Published on Jul 24, 2013, Changping, C., Yanshuo, W., Rong, L., Jianxin, Z., Yahui, h., “Sodium alginate-sodium carboxymethylcellulose-chitosan wound dressing and preparation method thereof”, Patent No. CN104069537 A, Published on Oct 1, 2014; Hao, C., Chuanrong, L., Qiujie, Y., Yiming, Jihu, W., Chen, X., Jincheng, W., Jianzhong, f.,”Medicinal spongy slow release material as well as preparation method and application thereof”, Patent No. CN102824303 A, Published on Dec 19, 2012). Blended gel matrix was also evaluated for dual functionality. Alginate-gelatin blend without voids was studied in controlled release and tissue reconstruction recently (Zhang and Li 2010, Yumin, D., Li, F., Baozhong, Z., Qun, W., “Blended fiber of sodium alginate/glutin, preparation method and application thereof”, Patent No. CN1296534 C, Published on Jan 24, 2007; Palempalli, S., Jayanti, V.R., “Controlled release formulations of anti-tubercular drugs using different biodegradable natural polymers”, Application No. : 3305/CHE/2008, Publication on July 9,2010; BAES, E., WOLD, I.M., “Gelatin/alginate delayed release capsules comprising omega-3 fatty acids, and methods and uses thereof”, US Patent No. US20150004226 A1, Published on Jan 1, 2015 Baes and Wold 2015).

As apparent from the state of the art that while there are various methods prevalent in the state of the art for making a porous scaffolds, blend or composite scaffolds with embedded voids excellent strength and desired macro porosity with tunable release kinetics was never attempted and hence it is the need of the day to provide for such hydrogel scaffolds for effective use in drug delivery and tissue reconstruction.

OBJECTS OF THE INVENTION
It is thus the primary object of the present invention to provide for hydrogel scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength.

It is another object of the present invention to provide for said hydrogel scaffold with better absorptivity and mechanical strength, controlled macroporosity through introduction of a series of self- aligned voids using a fluidic arrangement such that the voids are sustained till the point of gelation to favour controlled macroporosity.

Yet another object of the present invention is to provide for said hydrogel scaffold with augmented intrinsic porosity of the gel matrix based on retention of the pore structure during solvent removal phase (lyophilization).

Another object of the present invention is to provide for said hydrogel scaffold with tunable release rate of the actives through said scaffold based on the use of chitosan layer over the super absorbing and quick releasing structure of biopolymer such as alginate.

Yet another object of the present invention is to provide for said hydrogel scaffold with controlled macroporosity and spatial distribution, and method of its preparation which is far inexpensive as compared to the conventional methods e.g., emulsion vacuum drying, gas foaming or thermal phase separation which method would also not require any exposure to any thermal or chemical treatment.
SUMMARY OF THE INVENTION

Thus according to the basic aspect of the present invention there is provided a hydrogel scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength.

Preferably in said hydrogel scaffold said cross-linked rigid biopolymer include alginate having ordered structure of self-assembled bubble based embedded voids of desired macroporosity, said voids formed of surfactant sustained bubble induced in said alginate.

More preferably, said hydrogel scaffold comprises single phase blended or layered composite scaffold of at least two or more biopolymer including self-assembled multilayer voids with macroporosity of said voids in the order of at least 100 µm.

According to another preferred aspect of the present invention said hydrogel scaffold comprises controlled macroporosity including series of self-aligned voids.

According to yet another preferred aspect of the present invention said hydrogel scaffold comprises biopolymers selected from alginate, gelatin, chitosan or mixtures thereof.

According to another preferred aspect of the present invention in said hydrogel scaffold said single phase blended scaffold comprises blend of two or more biopolymers including said self-aligned voids and layered scaffold includes sandwich type composite scaffold involving plurality of biopolymers layered atop one another including said self-aligned voids.

Advantageously, said hydrogel scaffold of the present invention includes scaffold for drug delivery or tissue reconstruction with anyone or more induced characteristics selected from i) strength to sustain the loading on the scaffold until degradation occurs, ii) diffusion of biological entities in the scaffold at a suitable rate, iii) an induced desired macroporosity allowing cell colonization and vessel formation, to develop further the elastomeric qualities with tunable compressive moduli and strength, and to balance rate of resorption with the rate of tissue conduction.

According to another preferred aspect of the present invention said hydrogel scaffold is a blend scaffold comprising alginate and gelatin blend in the ratio of 1 to 4 : 1 to 4 preferably 3:2 with embedded voids, absorptivity of 1630 to 3400 preferably about 3314.28% and mechanical strength of 0.003 to 0.05 preferably about 0.033 MPa as compared to void free alginate and gelatin scaffold having absorptivity at 2439.31%.

According to another aspect of the present invention a process for the preparation of hydrogel scaffold is provided comprising:
i) providing for biopolymer combination in blended and/or layered form;
ii) said biopolymer combination being obtained of atleast one polymer having tunable structure of induced multilayer voids of selectively desired anyone or more of macroporosity, elasticity and strength involving combination of ordered structure of self-assembled bubble based embedded voids of desired macroporosity obtained of surfactant sustained bubbles of aqueous suspension of said biopolymer prior to cross-linking and gelation.

According to another preferred aspect of the present invention said process for the preparation of hydrogel scaffold includes blend or layered composite scaffold of cross-linked rigid biopolymer gel free of any chemical or thermal treatment comprising the steps of
a. providing an aqueous solution/suspension of biopolymer/s as homogeneous single phase solution free of any bubble;
b. adding surfactant to said homogeneous aqueous solution/suspension of biopolymer/s to suspend the mixture of biopolymers in a single phase;
c. introducing and embedding bubbles through a fluidic co-flow device to provide for self aligned bubble based polymeric film;
d. optionally, layering a biopolymer on said polymeric film to generate a sandwich type structure;
e. crosslinking and gelation followed by vacuum drying or optionally lyophilization to attain said hydrogel scaffold including blend or layered composite scaffold with desired macroporosity and intrinsic porosity.

Preferably in said process for the preparation of hydrogel scaffold of a single biopolymer said step (a) involves providing an aqueous solution of single biopolymer;
wherein said step (d) involves crosslinking with CaCl2 solution preferably a 4 wt% CaCl2 solution to obtain hydrogel scaffold of desired macroporosity and intrinsic porosity.

Preferably in said process for the preparation of hydrogel scaffold as blended composite scaffold said process comprise the steps of
(a) providing a blended aqueous suspension of at least two or more biopolymers as single phase homogeneous solution free of any bubble preferably aqueous suspension of alginate and gelatin;
(b) adding surfactant preferably pluronic F-127 to said homogeneous aqueous suspension to retain it as a single phase suspension;
(c) introducing and embedding bubbles through said fluidic co-flow device providing for self aligned bubble based polymeric film preferably at liquid/ aqueous suspension flow rate of 5 mL/mim and a gas flow rate at 1 mL/min wherein said introduced bubbles could be sustained prior to crosslinking and gelation;
(d) crosslinking and gelling by crosslinking agent preferably 4 wt% CaCl2 solution followed by vacuum or freeze drying to obtain said blended composite scaffold therefrom of desired macroporosity.

Preferably in said process for the preparation of hydrogel scaffold as layered composite scaffold said process comprise the steps of
(a) providing an aqueous solution of a biopolymer as a homogeneous solution free of any bubble preferably aqueous solution of alginate;
(b) adding surfactant preferably pluronic F-127 to said homogeneous aqueous solution of alginate;
(c) introducing and embedding bubbles through said fluidic co-flow device providing for self aligned bubble based polymeric alginate film preferably at aqueous solution flow rate of 5 mL/min and a gas flow rate at 1 mL/min wherein said introduced bubbles was sustained to provide for voids in the alginate gel that was preferably vacuum dried preferably for 2 hours to remove unbound moisture or the surface moisture from the polymeric gel film attained;
(d) optionally, layering a biopolymer preferably chitosan on both the sides of said polymeric alginate gel film prior to the development of gel structure of chitosan to generate a sandwich type composite structure;
(e) adding formaldehyde solution to chitosan solution for crosslinking and gelation followed by vacuum drying and lyophilization to attain said hydrogel scaffold as layered composite scaffold with desired macroporosity and intrinsic porosity.

Preferably in said process for the preparation of said hydrogel scaffold said step (d) involves layering preferably about 2 wt% chitosan solution in 0.2 N Acetic acid solution was layered on 2 wt% alginate gel with voids;
wherein in step (e) the composite structure film was vacuum dried at 40°C for about1 hour and was thereafter dried in a freeze drier for about 17 hours.

According to another preferred aspect of the present invention in said process for the preparation of hydrogel scaffold said step (e) involves lyophilization preferably in a freeze drier conducted in three phases wherein in the first phase, the temperature of the scaffold was reduced to - 30°C to freeze the moisture present in the scaffolds without disturbing the gel strands, wherein in the second phase (sublimation) a vacuum of about 0.09 mbar (absolute) was imposed on the scaffold and the temperature increased to 0°C with the increase of temperature from -30°C following a ramp of 10°C/ 60 min, and a soak at -20°C, -10°C, and 0°C for 45 minutes, and wherein in the third phase (secondary drying) the temperature was increased to upto about 40°C through a similar ramp and soak sequence by maintain the vacuum all along favouring augmentation of intrinsic porosity of the gel matrix based hydrogel scaffold by retention of the pore structure during lyophilization.
BRIEF DESCRIPTION OF FIGURES
Figure 1: illustrates Schematic drawing of the “Orifice – in – Throat” arrangement;
Figure 2: illustrates Alginate-Gelatin bubble in a petridish of composition a) 1:1 b) 2:3 c) 3:2 and d) 1:1 multilayer;
Figure 3: illustrates SEM top and cross-sectional view images of Alginate – Gelatin, Alginate – Chitosan and Alginate–only scaffolds; (a) illustrates Alginate – Gelatin (3:2) without void; (b) illustrates Alginate – Gelatin (3:2) with void; (c) Alginate – Gelatin (2:3) without void; (d) illustrates Alginate – Gelatin (2:3) with void; (e) illustrates Alginate – Gelatin (1:1) without void; (f) illustrates Alginate – Gelatin (1:1) with void; (g) illustrates Alginate – Gelatin (1:1) with void Multilayer Cross-sectional view; (h) illustrates Alginate – Chitosan with void Cross-sectional view; (i) illustrates Alginate – Chitosan without void Cross-sectional view; (j) illustrates Alginate – only with void and without void top view; (k) illustrates microscopic cross-sectional image of Alginate scaffold;
Figure 4: illustrates release profile of Alginate – Gelatin (1:1) vacuum dried Scaffolds;
Figure 5: illustrates release profile of Alginate – Gelatin Freeze dried Scaffolds;
Figure 6: illustrates release profile of Alginate–Chitosan Freeze dried Scaffolds;
Figure 7: illustrates change in Young’s Modulus with respect to % Gelatin in blend scaffolds;
Figure 8: illustrates change in Ultimate Stress with respect to % Gelatin in blend scaffolds.

DETAILED DESCRIPTION OF THE INVENTION
As discussed hereinbefore the present invention provides for a hydrogel scaffold including single phase blended or layered composite scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength, which voids are formed of surfactant sustained bubble till the point of gelation that was induced in said biopolymer.
Said voids in regular arrangement advantageously provide elastomeric qualities in the final structure against compression loading. The bubbles were made in aqueous suspension of biopolymers. The bubbles rapidly self-aligned to provide an ordered structure that was retained by the surfactant till the point of gelation, added a priori. Further crosslinking of the polymer chains results in rigid gel with embedded voids. The method of the present invention potentially offers enhanced absorptivity, strength and better control of void size, as compared to the other existing conventional methods, e.g., emulsion vacuum drying, gas foaming or thermal phase separation. The matrix is not exposed to any chemical or thermal treatment by this method. The method is inexpensive when compared with the other precise techniques e.g., free from fabrication.

Biopolymers such as gelatin and chitosan are added to increase the mechanical strength of the dried film, while retaining the other properties such as, absorptivity. A fluidic device was utilized to introduce self-aligned series of bubbles that left microvoids in the final structure. One use of this structure can be in delivering biological agents to the host tissue, and in providing a three-dimensional support for matrix formation. The voids in regular arrangement may provide elastomeric qualities in the final structure against compression loading. The bubbles were made in aqueous suspension of biopolymers. The bubbles rapidly self-aligned to provide an ordered structure that was retained by the surfactant, added a priori. Further crosslinking of the polymer chains results in rigid gel with embedded voids. The method has the potential to offer enhanced absorptivity, strength and better control of void size, compared to the other conventional methods, e.g., emulsion vacuum drying, gas foaming or thermal phase separation. The matrix is not exposed to any chemical or thermal treatment by this method. The method is inexpensive when compared with the other precise techniques e.g., free form fabrication.
According to an embodiment of the present invention three compositions of blend gel scaffolds, prepared from alginate and gelatin are considered here. One composition of this blend gel (3:2) with embedded voids shows absorptivity of 3314.28%. The same in absence of voids was noted as 2439.31%. The mechanical strength of the blend gel scaffold was found as 0.033 MPa. The same for alginate-only scaffold of similar dimensions was 0.0106 MPa. A detailed comparison is provided in Table 1 hereunder.
Table – 1: Summarization of the blend scaffolds prepared from Alginate and Gelatin
Scaffolds tag Ratio
(Alginate : Gelatin) Blend Composition Drying method Presence of bubbles Thickness (mm) Pulling rate – 2 mm/min Absortivity (%)
Modulus of elasticity (MPa) Ultimate stress (MPa)
A 1:1 2% Alginate
2% Gelatin
2% Pluronic Vacuum drying With void 1 0.0941 0.003221 1631.463
B 1:1 2% Alginate
2% Gelatin
2% Pluronic Freeze drying With void 1 0.252± 0.09 0.049±0.006 1824.842
C 1:1 2% Alginate
2% Gelatin
2% Pluronic Vacuum drying Without void 1 0.140±0.0426 0.00525±0.00278 1114.29
D 1:1 2% Alginate
2% Gelatin
2% Pluronic Freeze drying Without void 1 0.346±0.055 0.0704±0.02 1244.646
E 3:2 2.4%Alginate
1.6%Gelatin
2% Pluronic Freeze drying With void 1 0.11±0.004 0.0201±0.003 3314.28
F 3:2 2.4%Alginate
1.6%Gelatin
2% Pluronic Freeze drying Without void 1 0.162±0.02 0.033±0.005 2439.31
G 2:3 1.6%Alginate
2.4%Gelatin
2% Pluronic Freeze drying With void 1 0.108±0.008 0.009±0.001 2817.919
H 2:3 1.6%Alginate
2.4%Gelatin
2% Pluronic Freeze drying Without void 1 0.141±0.02 0.027±0.02 2548.01

Another aspect of the present invention is the application of a chitosan layer over an alginate film. Here, the chitosan is used to support the alginate gel, and creating a membrane over the alginate to control the release rate. The voids in the alginate gel film were generated by the fluidic arrangement as before. The voids were meant to provide the necessary functions as envisaged for blended gel. The chitosan film offered a diffusivity, orders of magnitude less than that of alginate for a model solute e.g., Vitamin B12. Thus, the chitosan layer ensured a much slower release of solute, as demanded by the application. This advantage is over and above the strength that the chitosan layer offered to the composite structure. The adhesion between the two layers was found to be strong enough to bear the loading involved in uptake and release studies. The percentage absorptivity of Vitamin B12 is shown in Table 2 hereunder.
Table – 2: Summarization of the layered composite scaffolds prepared from Alginate and Chitosan
Scaffolds tag Layer Composition Drying method Presence of bubbles Thickness (mm) Absorptivity (%)
X 2% Alginate
2% Chitosan
2% Pluronic Freeze drying Alginate-With void
Chitosan- Without void 4.0 1668.534
Y 2% Alginate
2% Chitosan
2% Pluronic Freeze drying Alginate and
Chitosan- Without void 4.0 1213.142

Here, the alignment of voids in the blended films was studied under digital microscope. The film was crosslinked, and then lyophilized to remove water from the film. The uptake and release characteristics of the gel film were studied using Vitamin B12 as model solute and PBS buffer as the release medium. The mechanical strength of the swelled gel film was measured vis-a-vis uptake and release profile for gel films of different thickness, % gelatin, drying condition and presence of voids.
The present invention thus provides novel ways of tuning the properties of hydrogel scaffold for use in drug delivery and tissue reconstruction. The significant aspects of the methods of the present invention are as follows:

a. A composite scaffold with alginate and chitosan layers, crosslinked one over the other was prepared.
b. Foaming of alginate layer, and multiple layers of self-assembled bubbles, acting as main source of macroporosity in the composite scaffold was demonstrated.
c. Excellent absorption by the composite scaffold due to intrinsic porosity of the two gels, and the presence of multilayer of voids in the alginate layer was observed.
d. The rate of release from the composite scaffold is controlled by the diffusion through the chitosan layer. A release duration of hours to days for Vitamin B12 as model solute can be tuned by manipulating the thickness of the chitosan layer.
e. Excellent strength of the composite scaffold is rendered by the chitosan layer.
f. A fluidic device was used to generate a self-aligned void structure in the gel layer. Inside the fluidic device, comprising of two coaxial capillaries, a second level of split of bubble is orchestrated by squeezing the bubbles at the throat, and cause additional stretch and fold of the bubble prior to the detachment from the orifice. A bubble size on the order of the feature size of the throat could be generated, and self-aligned in the matrix, as the polymer gets crosslinked to form gel. The bubble size could further be controlled by changing the flow ratio. Any plugging from the lumped gel aggregates could be avoided by adopting this special fluidic arrangement. The self-alignment of bubbles in multilayer shows benefit of absorption by significantly large volume in a single embodiment, while increasing the strength of the film.
g. Biomedical engineering tissue grafts are used to mimic multiple functions. In this invension, two biopolymers were blended into a single phase, so that the final composition shows the benefit of both the biopolymers. Generally, it is difficult to keep the two biopolymers suspended together in water, and allow slow crosslinking. In most such cases, one or both separate out. In this invention, alginate and gelatin could be suspended together in a single phase in presence of a surfactant, and crosslinked to form gel.
h. The excellent absorption capacity of alginate gel, and the mechanical strength of gelatin or chitosan could be coupled in a blend or layered scaffold respectively together with embedded voids to show unique release kinetics.
i. The use of lyophilization to remove moisture from the composite structure induces high intrinsic porosity to the gel matrix.

Accordingly the advantages by way of the technical advancement of the present invention are as follows:
The scaffold for drug delivery or tissue reconstruction requires properties e.g., i) strength to sustain the loading on the scaffold until degradation occurs, ii) the diffusion of biological entities in the scaffold at a suitable rate, iii) an induced macroporosity on the order of 100 µm and above to allow cell colonization and vessel formation, to develop further the elastomeric qualities with tunable compressive moduli and strength, and to balance rate of resorption with the rate of tissue conduction. These properties are induced in the gel structure by two different ways. One method utilizes blending of two biopolymers. The other method uses layered composite of two biopolymers. The blending helps in tuning the strength and intrinsic diffusivity to the desired levels. The two biopolymer, chosen for blending are alginate and gelatin. The layered composite films was made with alginate and chitosan. The microporosity was introduced by injecting inert gas through a fluidic device.
The excellent absorption capacity of alginate gel, and the mechanical strength of gelatin are coupled in the blend scaffold utilizing their miscibility in the pH range of 5.5 to 6.3, in presence of added surfactant. In addition, the bubbles of size on the order of 100 µm were introduced using fluidic arrangement. The self alignment of bubbles within the gel structure provided uniform distribution of macroporosity. The orifice in throat arrangement, used here in making bubbles is a novel device from traditional glass blowing unit, where plugging from lumped gel aggregate can be avoided. The size of the bubbles is on the same order as the feature size of the orifice, and can be further tuned by changing the flow rates of the two phases. The present invention shows that self alignment of bubbles in single and multilayers. The use of freeze drying to remove water renders a high intrinsic porosity of the gel matrix.
Alginate and Chitosan together with embedded voids further selectively complement each other by showing high release rate, and low strength in swelled condition for alginate, and the reverse in case of chitosan to reveal unique release kinetics. However, it is difficult to keep the two biopolymers suspended in aqueous solution which the present invention overcomes. The present invention addresses the making of multilayered composite scaffold, which has benefit of both the polymers, and additionally the macroporosity in the alginate layer through induction of bubbles in multiple layers. The final scaffold of the present invention was found to have excellent strength based on the synergistic action of chitosan layer, and absorption capability provided by the alginate involving single or multilayer of voids. The diffusivity in chitosan is widely different from that of alginate. Thus, the two layers in tandem together with the embedded voids provide for the selectively tuned release kinetics. The other advantages such as, enhancement of intrinsic porosity by freeze drying etc. are applicable here as well. Thus the related advantages of the present invention are as follows:
1. Better absorptivity and mechanical strength in single embodiment.
2. Control of macroporosity through introduction of a series of self- aligned voids using a fluidic arrangement.
3. Augmentation of intrinsic porosity of the gel matrix by retaining the pore structure during solvent removal phase (lyophilization)
4. Tuning release rate through use of a chitosan layer over the super absorbing and quick releasing alginate structure.
5. Better control of macroporosity and its spatial distribution, compared to the conventional methods e.g., emulsion vacuum drying, gas foaming or thermal phase separation.
6. Inexpensive method
7. The method does not require exposure to any thermal or chemical treatment.

Examples:

Example 1: Method of preparing the hydrogel scaffold

Sodium alginate and Gelatin (100 bloom) solution of 6 wt% was prepared separately by dissolving an estimated amount of the polymers in double-distilled water at a constant stirring speed of 400 rpm on a magnetic stirrer for 24 hours. 6 wt% pluronic F-127 solution was prepared in double-distilled water, and was kept in a refrigerator for the complete dissolution. Here pluronic F-127 was used as a surfactant. The alginate and gelatin solutions were mixed on a magnetic stirrer at low rpm to avoid the bubble formation in the solution. The mixture was kept in a sonicator to obtain a homogeneous solution without any bubble. Then pluronic solution was added to the homogeneous mixture of alginate and gelatin on a magnetic stirrer. The co-flow device was used for embedding bubbles in the polymer film. A liquid flow rate of 5 mL/mim and a gas flow rate at 1 mL/min were used. 4 wt% CaCl2 solution was added drop-wise around the bubble. Eight such films were tagged with alphabetical codes A, B, C, D, E, F, G, and H respectively. Here, the composition was varied systematically. The details are in Table 1. In some cases, two identical scaffolds were submitted to different drying conditions. One was vacuum dried, and the other was freeze dried. The vacuum drying was conducted at 40°C and 50 torr for 6 hours.
Another type of scaffold, considered here is of sandwich-type structure. Here, the alginate film was kept in between two layers of chitosan. 2 wt% chitosan solution was prepared by dissolving an estimated amount of chitosan in 0.2 N Acetic acid solution at a constant stirring speed of 3000 rpm in a mechanical stirrer for 24 hours. The solution was filtered using a whatman-A type filter under vacuum to remove large suspended agglomerates. 4 wt% formaldehyde solution was prepared separately for crosslinking chitosan. Separately, 2 wt% alginate gel with voids was prepared as per the procedure described before. The alginate gel film was vacuum-dried for 2 hours to remove unbound moisture or the surface moisture. The crosslinking reaction was set by adding the formaldehyde solution to the chitosan solution. Before the gel structure developed in chitosan, the mixture was applied as a cover layer on the alginate film. The composite film was vacuum dried at 40°C for 1 hour. Next, the composite was flipped, and another layer of chitosan solution was deposited to the other side in a similar manner. Finally, the composite film was dried in a freeze drier for 17 hours. A second composite scaffold was made without any bubble in the alginate layer. The two scaffolds are referred with tags X and Y respectively in Table 2.
The lyophilization process in a freeze drier was conducted in three phases. In the first phase, the temperature of the scaffold was reduced to - 30°C. Here, moisture present in the scaffolds was frozen without disturbing the gel strands. In the second phase (sublimation), the vacuum of 0.09 mbar (absolute) was imposed on the scaffold, as the temperature was increased to 0°C. The increase of temperature from -30°C followed a ramp of 10°C/ 60 min, and a soak at -20°C, -10°C, and 0°C for 45 minutes. In the third phase (secondary drying), the temperature was increased all the way to 40°C through a similar ramp and soak sequence. The vacuum was maintained all along.
The bubble formation in the biopolymer film was conducted using “Orifice – in – Throat” arrangement, as shown in Figure 1. The bubble formation process was initiated through expansion of inner gas thread at the orifice. Next, the tip of this bubble entered into the constricted orifice at the end of the outer capillary. Here, the bubble was deformed to a dumbbell shape because of the squeeze at the outer tip, resulted due to higher pressure. Immediately, the gas string detached from the nozzle with a simultaneous split at the neck of the dumbbell. Thus, a series of two bubbles got detached from the flow device, and the meniscus got retracted. The procedure recurred and as a result, series of bubbles are formed.
The scaffolds were characterized extensively. The self-alignment of bubbles was observed under digital microscope, and the bubble size distributions were determined using an edge-detection algorithm. After the removal of water from the scaffolds, the images were acquired under scanning electron microscope. The strengths of the scaffolds in the swelled state were determined by subjecting them to varying tensile loads leading to a rupture. The ability of the dry scaffold to absorb a model drug (Vitamin B12) was measured gravimetrically using a below balance weighing facility. The release of vitamin B12 in PBS buffer was studied spectrophotometrically.
The viscosity data shows shear-thinning behavior for alginate, chitosan and alginate-gelatin mixture respectively. With increase in gelatin content, the viscosity of the mixture was found to increase.
Table – 3: Viscosity of the blend solutions

Blend composition Viscosity (cP)
at a shear rate of 1 s-1 pH
2:3 89.71 5.5
1:1 153.7 6.3
3:2 273.1 5.66

Figure 2 presents the images of the bubbles on a petridish. Three different composition of alginate and gelatin content was considered here. In every case, the gas flow rate of 1 ml/min, and the liquid flow rate of 5 ml/min were maintained. The bubbles were found monodisperse with average diameter of 1.2 mm. The self-alignment forced each bubble to get surrounded by six bubbles in a lattice structure.

Example 2: Structural details from SEM images

The SEM images after removal of water from the gel film are shown in Figure 3. A closer examination of the SEM images at low magnification shows flaky structure at the surface after lyophilization. With increase in gelatin content, the flaky structure disappeared, and the surface became much smoother. This is unlike the features, shown in Figure (3j) for vacuum dried alginate-only films. The blended gel did not show any formation of agglomerates within the gel matrix after water removal. This again verified that the polymers blend gels were homogenous, and no separation of polymer phases was observed in the gel matrix. The alignment and shape of voids remained intact during water removal for lyophilized and vacuum dried samples respectively. The Figures 3d and 3e indicate that the above observations can be extended to films with multi-layers of bubbles.
Another important observation from the SEM images is the effect of solvent removal on the morphology of the film, as observed in the cross-sectional views. The lyophilized films show microporosity at a length scale of around 10 µm. This value was found much smaller for the vacuum dried films. This aspect will be reviewed later in the report to analyze uptake and release of model solutes by the two types of gel films.
The SEM images of alginate chitosan layered composite are presented in Figures 3h through 3i. A layered structure is evident here with alginate layer squeezed in between two chitosan layer. The chitosan layers appeared much fudged up compared to alginate layer, possibly due to the use of lyophilization.
The cross-sectional views of alginate scaffold, acquired under a microscope are shown in Figure (3k). A cobweb-like structure indicates significant enhancement in porosity.

Example 3: Uptake and Release studies
Vitamin B12 is considered here as a model drug for uptake and release studies. 0.02 wt% Vitamin B12 solution was prepared in a PBS buffer solution of pH 7.4. The uptake of vitamin B12 was measured gravimetrically. The percentage absorptivity of the Vitamin B12 was obtained by the following equation.
% Absorption = (W_w-W_d)/W_d ×100 ….. (1)
Where, Wd is the initial weight of dry scaffold
Ww is the weight of the fully swelled scaffold.

The percentage absorptivity was found to be highest for blend scaffolds tagged as B, D, E and G. The blend scaffolds, tagged as A, C, F and H respectively showed lower absorptivity (Table 2). The freeze dried film showed more absorptivity than a simple vacuum dried film. The highest absorptivity of 3314.28% was observed for alginate-gelatin (3:2) blend scaffold, where water removal was by lyophilization. This is consistent with the porous structure of the lyophilized gel film that was observed in the cross-sectional view of the SEM images, discussed earlier.
The release experiments on the scaffolds, duly loaded with Vitamin B12 were conducted by dipping it in a 300ml PBS solution, and shaking at a constant speed on a rotary shaker. A sample of 5ml was withdrawn from the beaker (release media) at a time interval of 30 minutes. Simultaneously, a 5ml fresh PBS solution was added in the release media to maintain a constant volume of 300ml in the beaker. The concentrations of vitamin B12 in the withdrawn samples were measured using UV-vis spectrophotometer at 361 nm.
The release of vitamin B12 from the scaffolds are plotted in Figures 4, 5 and 6. From the scaffolds that were dried by vacuum only, the near complete release of Vitamin B12 was observed within 8 hours. This period was reduced to 4 hours for scaffolds that were freeze dried. This is another indication of the ease of transport of solutes in freeze dried gel network.

One purpose of adding gelatin to alginate was to improve the strength of the gel without sacrificing the absorptivity. The mechanical strength of blended gel film with and without voids respectively was measured using an ultimate testing machine. The sample size, used in these experiments are 10 mm × 36 mm and the displacement of the head was at a rate of 2 mm/min. Table 1 indicated the Young’s modulus and ultimate stress of the gel scaffold. It is evident here that the presence of gelatin added strength to the blended scaffold, as the strength of alginate-only scaffold of similar thickness (1 mm) without voids was found to be 0.0106 MPa.
A critical review of ultimate strength values for scaffolds of different alginate to gelatin mass ratios suggests that the strength did not increase monotonously with increase in gelatin concentration in the blend. Figures 7 and 8 present the Young’s modulus and ultimate strength as a function of gelatin concentration in the blend. For with and without voids respectively, the ultimate strength was found to decrease with addition of gelatin beyond a gelatin content of 50%. This review suggest that the structural integrity of the gel film with voids, as the matrix undergoes degradation in PBS buffer can be tuned by blending gelatin with alginate.

In the layered composite, the chitosan layer hindered the diffusion of Vitamin B12 out into the PBS buffer in release experiments. 80% of the total release was observed in first eight hour (Figure 6).The advantages here are manifold. Firstly, the thickness of the chitosan layer, and the choice of crosslinker for chitosan gel enable tuning of solute release rate. Secondly, the chitosan is a much stronger gel, and provide strength to the composite. Thirdly, the bubbles in alginate film can be generated in multiple layers, as they are encased by the chitosan films in the final product. Thus, the absorptivity can be increased significantly.

It is thus possible by way of the present advancement to provide for said hydrogel scaffold including single phase blended or layered composite scaffold comprising scaffold of rigidly cross-linked biopolymer having tunable structure of induced multilayer voids of desired macroporosity, elasticity and strength and thereby tunable release kinetics, which voids are formed of surfactant sustained bubble till the point of gelation induced in said biopolymer.
Said voids in regular arrangement advantageously provide elastomeric qualities in the final structure of the scaffold against compression loading. The bubbles were made in aqueous suspension of biopolymers. The bubbles rapidly self-aligned to provide an ordered structure that was retained by the surfactant till the point of gelation, added a priori. Further crosslinking of the polymer chains resulted in rigid gel with embedded voids. The method of the present invention thus potentially offers enhanced absorptivity, strength and better control of void size, as compared to the other existing conventional methods, e.g., emulsion vacuum drying, gas foaming or thermal phase separation. More advantageously, the matrix being not exposed to any chemical or thermal treatment renders said method inexpensive and cost-effective when compared with the other precise techniques e.g., free from fabrication.