Progression of fabrication of tissue scaffolds and hence conducting cell growth within

Presented by-Pritam Kishore Chakraborty, M.tech, MNDSMSE, IIEST Shibpur

WHAT THIS PRESENTATION IS ALL ABOUT-

ADVANCEMENTS OVER THE YEARS (1998-PRESENT) IN 3D BIOPRINTING AND ELECTROSPINNING FOR MAKING SCAFFOLDS AND SUCCESSFULLY CONDUCTING CELL

GROWTH WITHIN.

Project Overview:

WHAT IS 3D PRINTING

Image taken from paper by A. Park et al.

1998

• Paper presented by Park et al. [1] , where he synthesized substrates from Poly(L-lactide)(PLLA) & Poly(lactide-co-glycolate)(PLGA) using 3D printing technique for the first time, for preparing scaffolds.

• The substrate surface was modified using carbohydrate linkages facilitated by hydrophobic PEO-PPO-PEO tri-block copolymers.

• 3T3 fibroblasts and hepatocytes from mouse were used for preparing cell culture.

• Compared the results of cell adhesion(controlled) between tissues and substrates prepared by melt process and by 3D printing.

• Found superior results for 3D printed surface.

2002

• Lam et al.[2] successfully did Scaffold development using 3D printing with a unique blend of starch based polymer powders (cornstarch, dextran and gelatin), using a 3D printer developed and commercialized by Zcorp (Z402).

• The scaffold properties were characterised by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), porosity analysis and compression tests(Instron 4302 material testing system using a 1-kN load-cell).

• Results showed that natural polymers can act as good materials for tissue engineering by using 3D printing technique.

2002

• Cooke et al.[3] , showed Use of Stereolithography to Manufacture Critical-Sized 3D Biodegradable Scaffolds for Bone Ingrowth.

• The materials used in the experiment were poly(propylene fumarate) (PPF), diethyl fumarate (DEF) solvent & bisacylphosphine oxide(BAPO) photoiniator.

• The primary objective of the experiments was to investigate if it is possible to crosslink a (PPF) resin mixed with a suitable photoinitiator with the use of stereolithographic process, and to generate a scaffold with highly accurate control of the external surface and internal geometry that was of similar size and complexity to scaffolds useful for human bone tissue engineering.

• They were somewhat successful in achieving about 90% porosity.

2003

• Pfister et al.[4], did experimentation on Biofunctional Rapid Prototyping for Tissue-Engineering Applications and compared results between 3D Bioplotting versus 3D Printing.

• Two important rapid-prototyping technologies (3D Printing and 3D Bioplotting) were compared with respect to the computer-aided design and free-form fabrication of biodegradable polyurethane scaffolds meeting the demands of tissue engineering applications.

• The scaffolds were characterized with X-ray microtomography,scanning electron microscopy, and mechanical testing.

• 3D Bioplotting here showed superior results as shown in the table below.

2003

2004

• Krebsbach et al.[5] , developed methods for casting scaffolds that contain designed and controlled locally and globally porous internal architectures.

2004

• Lee et al.[6] demonstrated Scaffold fabrication by indirect three-dimensional printing.

• This study describes an indirect 3DP protocol, where molds are printed and the final materials are cast into the mould cavity to overcome the limitations of the direct technique.

• The direct 3DP approach incurs several limitations on scaffold structures, 3DP nozzles deposit small droplets with low KE in order to maximize resolution. Incomplete penetration will reduce layer-to-layer connectivity, and lead to lamination defects. Larger or faster droplets can penetrate thicker layers, at the expense of print resolution.

• A second limitation of the direct 3DP approach is shape complexity when the powder material (e.g. degradable polymer) demands the use of organic solvents as liquid binders, because solvents will also dissolve most commercially available drop-on-demand printhead subsystems.

2004

• Finally, the direct 3DP approach for biodegradable polymers and solvents requires custom machines, proprietary control software, and extensive operator expertise.

• Using the indirect 3DP technology, we were able to fabricate scaffolds with large pore sizes as well as scaffolds with fine features, using commercially available 3D systems. The scaffolds made by indirect 3DP supported cell growth in culture.

2005

• Leukers et al.[7], Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing using spray-dried HA-granulate containing polymeric additives to improve bonding and flowability. A water soluble polymer blend (Schelofix) was used as binder.

• Seitz et al.[8] did similar work on Three-Dimensional Printing of Porous Ceramic Scaffolds for Bone Tissue Engineering.

• Zhang et al.[9] in the same year did work on Microrobotics and MEMS-Based Fabrication Techniques for Scaffold-Based Tissue Engineering using rapid prototyping technique.

2007

• Duan et al.[10] synthesised Hybrid nanofibrous membranes of PLGA/chitosan fabricated via an electrospinning array.Hybrid nanofibrous membranes of poly(lactic co-glycolic acid) (PLGA) and chitosan with different chitosan amounts (32.3, 62.7, and 86.5%) were fabricated via a specially designed electrospinning setup consisting of two sets of separate syringe pumps and power supplies.

• The structure, mechanical properties, water uptake,and cytocompatibilities of the nanofibrous membranes were investigated by scanning electron microscopy, tensile testing,incubation in phosphate buffer solution, and human embryo skin fibroblasts culturing.

• Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid PLGA/chitosan membranes after chitosan crosslinking exhibited good mechanical and water absorption properties. The cytocompatibility of hybrid PLGA/chitosan membranes was better than that of the electrospun PLGA membrane. The electrospun hybrid nanofibrous membranes of PLGA and chitosan appear to be promising for skin tissue engineering.

2007

• Sun et al.[11], along with his team worked on Development of a 3D Cell Culture System for Investigating Cell Interactions With Electrospun Fibers.

• Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid(PLLA), with a broad range of diameters were constructed in a specifically developed 3D cell culture system.

• When fibroblasts were introduced to freestanding fibers, and encouraged to ‘‘walk the plank,’’ a minimum fiber diameter of 10 mm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 mm was also observed to be the maximum gap that could be bridged by cell aggregates—a behavior not seen in conventional 2D culture.

2008

• Blackwood et al.[12], presented their paper on “Development of biodegradable electrospun scaffolds for dermal replacement” based on their work.

• We examined a total of six experimental electrospun polymer scaffolds: (1) poly-L-lactide (PLLA); (2) PLLA +10% oligolactide; (3) PLLA + rhodamine and (4–6) three poly(D,L)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months.

• In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsuleformation around the periphery.

2008

• Jeong et al.[13], presented their paper “Electrospun gelatin/poly(L-lactideco-ε-caprolactone) nanofibers for mechanically functional tissue engineering scaffolds”.

• Blends of gelatin and poly(L-lactide-co-ε-caprolactone) (PLCL) (blending ratio: 0, 30, 70 and 100 wt% gelatin to PLCL) were electrospun to prepare nano-structured non-woven fibers for the development of mechanically functional engineered skin grafts.

• When seeded with human primary dermal fibroblasts and keratinocytes on the nanofibers, both initial cell adhesion and proliferation rate increased as a function of the gelatin content in the blend.

2013

• Lin et al.[14] worked on , “Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold”.

• Among the biomaterials commonly used for making nanofibrous wound dressings, chitosan and its derivatives are efficient. Chitosan is biodegradable, biocompatible, and can accelerate the tensile strength of wounds by speeding the fibroblastic synthesis of collagen in the first few days of wound healing and promote tissue regeneration.

• Here the chitosan was strengthened with pectin as a crosslinnker and Polyvinyl Alcohol was mixed to facilitate electrospinning of the mixture.

• The novel pectin cross-linked chitosan electrospun scaffold was stronger and less likely to break than a plain chitosan electrospun scaffold.

2014

• Chang et al.[15], worked on “A three-dimensional dual-layer nano/microfibrous structure of electrospun chitosan/poly(D,L-lactide) membrane for the improvement of cytocompatibility”.

• In this work, the electrospinning process incorporated with rotating control was introduced to prepare the random PDLLA microfibrous matrix covered with aligned chitosan nanofibers, providing an increased specific interface area to promote cell attachment, migration and infiltration.

• The results suggest that the dual-layer chitosan/PDLLA structure can mimic the interface of natural extracellular matrices and has the potential for 3-D tissue-engineering applications.

2014

• Gu et al.[16],presented his work “Adiposed-derived stem cells seeded on PLCL/P123 eletrospun nanofibrous scaffold enhance wound healing”.

• The aim of this study was to evaluate the wound healing of adipose-derived stem cells (ADSCs) seeded on electrospinning poly (L-lactide-co-ε-caprolactone)/poloxamer (PLCL/P123) scaffolds and the mechanisms using a rat skin tissue injury model.

• The ADSCs seeded on PLCL/P123 scaffolds could promote wound healing and have the potential to become a new therapeutic alternative material for skin tissue engineering.

2014

• Lou et al.[17] presented their paper , “Bi-Layer Scaffold of Chitosan/PCL-Nanofibrous Mat and PLLA-Microporous Disc for Skin Tissue Engineering”.

• The cell proliferation was found to be greatest in co-culture on bi-layer scaffolds.

• The gene and protein expression analyses further confirmed can the bi-layer scaffold provided a micro-environment similar to those present in the native extracellular matrix during initial wound healing

2015

• Sridhar et al.[18] , presented his paper “Improved regeneration potential of fibroblasts using ascorbic acid-blended nanofibrous scaffolds”.

• PLACL/SF/TCH/AA nanofibrous scaffolds were fabricated by electrospinning and characterized for fiber morphology, membrane porosity, wettability, and significant subchains using FTIR spectroscopy for culturing human-derived dermal fibroblasts.

• He showed that introduction of naturally secreting compounds (AA) by the cells into the nanofibrous scaffolds will favour cell’s microenvironment and eventually leads to complete tissue regeneration.

2015

• Steffens et al.[19], presented their paper , “Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute”.

• The study aimed to produce a cutaneous substitute, bringing together stem cells (mesenchymal stem cells) and keratinocytes, and an electrospun biomaterial.

• Three groups of scaffolds were studied: group 1,poly- dl -lactic acid (PDLLA); group 2, hydrolyzed PDLLA (PDLLA/NaOH) and group 3, PDLLA/Lam – a PDLLA/NaOH scaffold linked to laminin protein. They were characterized by physicochemical and biological parameters.

• Group 3 showed the best results concerning cell adhesion and viability

Remarks

• PLLA, Chitosan and cellulose derivatives are the most widely used matrix for preparing scaffolds.

• More and more research is getting focused on natural sources as alternatives for crosslinkers and ion sources for both 3D printing as well as electrospinning respectively.

• A combination of both can yield better results than comparative study among them as they can give combined effect of strength and elasticity, cellular adhesion,porosity and tissue regeneration.

Conclusion

• Finalizing on the materials especially the natural sources, is the most important aspect for tissue scaffold engineering as the healing properties are directly dependent on them.

Works Cited

1. Park A, Wu B, Griffith LG. Integration of surface modification and 3D fabrication techniques to prepare patterned poly (L-lactide) substrates allowing regionally selective cell adhesion. Journal of Biomaterials Science, Polymer Edition. 1998 Jan 1;9(2):89-110.

2. Lam CX, Mo XM, Teoh SH, Hutmacher DW. Scaffold development using 3D printing with a starch-based polymer. Materials Science and Engineering: C. 2002 May 31;20(1):49-56.

3. Cooke MN, Fisher JP, Dean D, Rimnac C, Mikos AG. Use of stereolithography to manufacture critical‐sized 3D biodegradable scaffolds for bone ingrowth. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2003 Feb 15;64(2):65-9.

4. Pfister A, Landers R, Laib A, Hübner U, Schmelzeisen R, Mülhaupt R. Biofunctional rapid prototyping for tissue‐engineering applications: 3D bioplotting versus 3D printing. Journal of Polymer Science Part A: Polymer Chemistry. 2004 Feb 1;42(3):624-38.

5. Taboas JM, Maddox RD, Krebsbach PH, Hollister SJ. Indirect solid free form fabrication of local and global porous, biomimetic and composite 3D polymer-ceramic scaffolds. Biomaterials. 2003 Jan 31;24(1):181-94.

6. Lee M, Dunn JC, Wu BM. Scaffold fabrication by indirect three-dimensional printing. Biomaterials. 2005 Jul 31;26(20):4281-9.

7. Leukers B, Gülkan H, Irsen SH, Milz S, Tille C, Schieker M, Seitz H. Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing. Journal of Materials Science: Materials in Medicine. 2005 Dec 1;16(12):1121-4.

Works Cited

8. Seitz H, Rieder W, Irsen S, Leukers B, Tille C. Three‐dimensional printing of porous ceramic scaffolds for bone tissue engineering. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2005 Aug 1;74(2):782-8.

9. Zhang H, Hutmacher DW, Chollet F, Poo AN, Burdet E. Microrobotics and MEMS‐based fabrication techniques for scaffold‐based tissue engineering. Macromolecular bioscience. 2005 Jun 24;5(6):477-89.

10. Duan B, Wu L, Yuan X, Hu Z, Li X, Zhang Y, Yao K, Wang M. Hybrid nanofibrous membranes of PLGA/chitosan fabricated via an electrospinning array. Journal of biomedical materials research part A. 2007 Dec 1;83(3):868-78.

11. Sun T, Norton D, McKean RJ, Haycock JW, Ryan AJ, MacNeil S. Development of a 3D cell culture system for investigating cell interactions with electrospun fibers. Biotechnology and bioengineering. 2007 Aug 1;97(5):1318-28.

12. Blackwood KA, McKean R, Canton I, Freeman CO, Franklin KL, Cole D, Brook I, Farthing P, Rimmer S, Haycock JW, Ryan AJ. Development of biodegradable electrospun scaffolds for dermal replacement. Biomaterials. 2008 Jul 31;29(21):3091-104.

13. Jeong SI, Lee AY, Lee YM, Shin H. Electrospun gelatin/poly (L-lactide-co-ε-caprolactone) nanofibers for mechanically functional tissue-engineering scaffolds. Journal of Biomaterials Science, Polymer Edition. 2008 Jan 1;19(3):339-57.

14. Lin HY, Chen HH, Chang SH, Ni TS. Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold. Journal of Biomaterials Science, Polymer Edition. 2013 Mar 1;24(4):470-84.

Works Cited

15. Chen SH, Chang Y, Lee KR, Lai JY. A three-dimensional dual-layer nano/microfibrous structure of electrospun chitosan/poly (d, l-lactide) membrane for the improvement of cytocompatibility. Journal of Membrane Science. 2014 Jan 15;450:224-34.

16. Gu J, Liu N, Yang X, Feng Z, Qi F. Adiposed-derived stem cells seeded on PLCL/P123 eletrospun nanofibrous scaffold enhance wound healing. Biomedical Materials. 2014 May 20;9(3):035012.

17. Lou T, Leung M, Wang X, Chang JY, Tsao CT, Sham JG, Edmondson D, Zhang M. Bi-layer scaffold of chitosan/PCL-nanofibrous mat and PLLA-microporous disc for skin tissue engineering. Journal of biomedical nanotechnology. 2014 Jun 1;10(6):1105-13.

18. Sridhar S, Venugopal JR, Ramakrishna S. Improved regeneration potential of fibroblasts using ascorbic acid‐blended nanofibrous scaffolds. Journal of Biomedical Materials Research Part A. 2015 Nov 1;103(11):3431-40.

19. Steffens D, Mathor MB, Santi BT, Luco DP, Pranke P. Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute. Regenerative medicine. 2015 Nov;10(8):975-87.

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