custom 3d scaffolds for regenerative medicine applications · audie l. murphy memorial va hospital...

Audie L. Murphy Memorial VA Hospital

Eric M. Brey, Ph.D.

South Texas Veterans Health Care System

University of Texas at San Antonio

Custom 3D Scaffolds for

Regenerative Medicine Applications


Craniofacial Defects

Craniomaxillofacial reconstruction often requires complex, multistage surgical procedures.

There is a critical need for improved methods for reconstruction of complex skeletal defects.

Craniomaxillofacial injury is the primary unfitting condition in many soldiers evacuated from current conflicts.

Sutradahar et al., PNAS, 2010


Tissue Engineering

“Tissue engineering is the

application of the principles and

methods of engineering and the life

sciences toward the fundamental

understanding of structure-

formation relationships in normal

and pathological mammalian tissues

and the development of biological

substrates to restore, maintain or

improve functions.”

Skalak and Fox (eds.) , Tissue Engineering, Alan Liss 1, 1, 1995

Sutradahar et al., PNAS, 2010


Akar et al., Tissue Engineering Part B, 2018

Approach

Image the defect

Identify the structure required

Generate PMMA chambers

Load tissue engineering strategy

Implant against the periosteum

Harvest

Transfer to the defect


in vivo bioreactor – growth of vascularized bone within the body by implantation of a molded chamber against

the periosteum (vasculogenic, osteogenic) in an uncompromised location

Brey et al., Plast Rec Surg, 2007; Cheng et al, Tissue Eng, 2005, Plast Rec Surg, 2006, 2009.


Periosteum guided prefabrication using morcellilzed bone graft can lead to the growth of vascularized bone of clinical size and volume

Brey et al., Plast Rec Surg, 2007; Cheng et al, Tissue Eng, 2005, Plast Rec Surg, 2006, 2009.


Design a tissue engineering scaffold that stimulates directed vascularization into the material while

maintaining tissue volume.

z


• Biocompatible, resistant to protein and cell

adhesion

• Incorporation of biofunctionality into hydrogels

by immobilization of bioactive derivatives of

photopolymerizable monomers

• Introduction of poly(L-lactic acid) units generate

copolymers that are degradable by hydrolysis

Poly(ethylene glycol) hydrogels


Pore Size• Porosity allowed invasion in the absence

of degradation.

• Vessel invasion varies with pore size in vitro and in vivo

• Very little vessel invasion observed in pore size < 50 µm

Chiu et al., Biomaterials. 2011

Vessel invasion depth

Chiu et al., Tissue Eng Part C Methods. 2010


Fibrin loaded pores

Fibrin loaded in the pores throughout the scaffold volume

Fibrin stimulated vascular ingrowth in a dose dependent manner

Jiang et al., Tissue Eng Part A. 2013

Vessel density

Audie L. Murphy Memorial VA HospitalChiu et al. PLoS One, 2013; Chiu et al., J Fluoresc 2012

• Introduction of poly(L-lactic acid) units into PEG hydrogels to generate copolymers that are degradable by hydrolysis

• By varying the ratio of PEG-DA to PEG-PLLA-DA we can generate porous hydrogels with varying degradation times

• The hydrogels maintain their pore size and structure during degradation

Degradation

Pore size

Degradation time


Gradient Scaffold Preparation

25% PEG-DA (Mw ≈8000) Fibrinogen within the pores

300-500 µm pores PLGA microspheres in 10 % PEG-PLLA-DA

10 mm

4 m

m

•PEG-DA: Polyethylene glycol diacrylate•PLGA : Poly(lactic-co-glycolic acid)•PDGF-BB: Platelet-derived growth factor •PEG-PLLA-DA: Poly (ethylene glycol)-co-(L-lactic acid) diacrylate

Distal layer

Porous Hydrogel

A B CC


NIR Imaging

Akar et al., Biomaterials 2015


Invasion and Vascularization

Green: TissueRed: Blood vessels

Week 1 Week 3 Week 6

100 µm 100 µm 100 µm

100 µm100 µm100 µm

Blank

200 ngPDGF-

BB

Akar et al., Biomaterials 2015

Vessel density

Invasion Depth


• Hydroxyapatite (HA) and β-tri-calcium phosphate (β-TCP) ceramic particles were incorporated into the composite scaffolds • Varying weight ratios (70:30, 50:50,

30:70% HA: β-TCP).

• The presence of ceramic particles within scaffolds significantly extended the degradation time.

• HA and TCP ceramics to this scaffold system enhanced bone formation.

Ceramic Composites


Porcine Model

10

m

m

17.5:7.5 % PEG-PLLA-DA:PEG-DA/Fibrin

• Polymer: Ceramic = 2:1 (w/w)

70:30%: HA:TCP

200 ng PDGF-BB

PMMA chamber

Cuff (adhesion)

Gradient scaffold


Evaluation


Modeling• Agent based model of

angiogenesis in porous scaffolds

• Results predicted experimental results for the influence of pore architecture

• Invasion depends

• Pore size

• Porosity

• Interconnectivity

• Size distribution

Mehdizadeh et al., Biomaterials 2013


3D Printing

Wang et al., Advanced Materials. 2015

• Precise control of internal and external architecture


Large Animal Defect Model

• Porcine mandibular defect model

• Evaluating surgical transfer of functional engineered bone formed in the in vivo bioreactor

Imaging courtesy of John Decker


Conclusions

• The in vivo bioreactor can result in vascularized bone of clinicalvolume and customized shape.

• A cell free approach requires optimization of structure, degradationkinetics and spatio-temporal release of growth factors.

• We are currently applying our approaches for engineeringvascularized bone in a large animal, clinically-translatable model of anin vivo bioreactor.


Acknowledgements - Funding

Veterans Administration (5 I01 BX000418-06 ); NIH (5R01EB020604-02, 1R01AR061460-01);

AHA (Innovators Research Grant); NSF (IIS-1126771, CBET-1263994, EEC-1157041, DSES-1635661)


Army Institute of Surgical Research

John Decker, D.M.D., M.S.

Erik Weitzel, M.D.

Chang Gung Memorial Hospital

Ming-Huei Cheng, M.D.

Hui-Yi Hsiao, Ph.D.

Illinois Institute of Technology

Ali Cinar, Ph.D.

Elisabeth Hildt, Ph.D.

Georgia Papavasiliou, Ph.D.

Rice University

Tony Mikos, Ph.D.

Wake Forest Institute for Regenerative Medicine

Emanuel C. Opara, Ph.D.

Washington University in St. Louis

Mark Anastasio, Ph.D.

University of Maryland – College Park

John Fisher, Ph.D.

University of Chicago

Ronald Cohen, M.D.

University of Belgrade

Lada Zivcovic, Ph.D.

University of Los Andes

Juan Carlos Briceno, Ph.D.

University of Texas at San Antonio

Gabriela Romero, Ph.D.

Amina Qutub, Ph.D.

Chris Rathbone, Ph.D.

Acknowledgements - Collaborators


Jacob Brown

Brenda Carrillo

Madeleine Farrer

Maria Gonzalez Porras, Ph.D.

Christina Jones

Kelly Langert, Ph.D.

Paola Lerma

Samantha Mann

Meritxell Martinez

Favour Obuseh

Binita Shrestha, Ph.D.

Katerina Stojkova

Feipeng Yang

Acknowledgements - Lab

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