Project Page - Adam Hacking - High throughput in vivo array

Created June 25 2008

Note this is a work in progress please feel free to add comments and suggestions

Many thanks Adam

ACTION PLAN

PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)

GENERATION OF A HIGH THROUGHPUT SYSTEM FOR IN VIVO EVALUATION OF BIOMATERIALS AND SOLUBLE FACTORS

A) Background

B) Specific Aims

C) General Approach

D) Design and Fabrication of in vivo chip

E) Design Pitfalls and alternatives for in vivo chip

F) Candidate Materials for Evaluation

G) Analytical approach Pre-Implantation

H) Analytical approach Post-Implantation

I) Future Directions

Background: Optimization of biomaterials for in vivo response is a complex task. In many instances the complex in vivo environment, such as that experienced during wound healing, bone formation or angiogenesis cannot adequately be modeled in vitro. As a result, testing and evaluation of many different biomaterials is generally carried out in vivo. However, in vivo testing is complicated by inter-animal differences and the practical limitations on the number of materials that can be evaluated. In cases where there are many materials and multiple permutations of growth factors, in vivo evaluation can become impractical. In such cases a high throughput in vivo array for testing biomaterials, cells and growth factors may be of benefit. The aim of this project is to develop system to evaluate biomaterials in vivo in a high throughput manner.

A) SPECIFIC AIMS

AIM 1 - A) Develop an in vivo array for evaluating osteoblast response to gels and hydrogels

B) Develop an in vivo array for evaluating osteoblast response to growth factors

AIM 2 - A) Develop an in vivo array for evaluating endothelial response to gels and hydrogels

B) Develop an in vivo array for evaluating endothelial response to growth factors

B) GENERAL APPROACH

Aim 1- In vivo model to assess potential for bone formation

Arrays will be implanted under the cranial periosteum of the rat. (Flat surface)

Anticipated result - ingress of osteogenic cells (early)

- formation of mineralized tissue

Aim 2 - In vivo model to assess potential for vascularization

Arrays will be implanted subcutaneously on the muscle on the back of the rat - (Large muscle access)

Anticipated result - ingress of vasculogenic cells (early)

- formation of vascular tissue

C) DESIGN AND FABRICATION OF IN VIVO CHIP

Q - Registration and record keeping of wells ?

Arrays fabricated from PEG and bonded to glass (Lifeng)

Pro - Fabrication in hand, facilitates loading, matches in vitro work,

Con -unknown long term performance in vivo, need to bond to materials that can be sutured in place i.e. plastic backing, wells may be too close, Leaching of materials from wells?.

Arrays fabricated from Methylmethacrylate (MMA)

Pro - Fabrication not a problem, no bonding to other substrates, good bio-compatibility, can suture in place, fix to skull, good optical clarity no well-well leaching

Con - Not same fabrication or in vitro environment as previous work (Lifeng)

Well Diameter - justification for 400-500 um diameter wells

Concepts for Biomaterial Assessment Array (Based on Lifeng's Chip)

Soft Tissue Array

2 chips per rat

Fix to muscle (good vascular ingress and homogeneity) with Nylon Sutures

Array face towards muscle, back (grey) towards skin

Implant for 7,14,21 days

Osseous Tissue Array

1 chip per rat

Fix back (grey) to skull with SuperGlue

Narrower since skull is narrow

Grey side fixed to bone, array faces periosteum

D) DESIGN CONCERNS FOR IN VIVO CHIP

E) CANDIDATE MATERIALS FOR EVALUATION

F) ANALYTICAL APPROACH (PRE-IMPLANTATION)

G) ANALYTICAL APPROACH (POST-IMPLANTATION)

What will be considered a successful test ?

H) FUTURE DIRECTIONS

• • Test materials in models of human disease - Bone (OP, OA).

  • Smaller chips for testing in mice? Validation in a mouse model.

  • Chips where we can capture information as well as examine response (see below).

  • Assess physiological processes -angiogenesis.

  • Entrap cancer cells / barrier membrane.

  • Real time monitoring of molecules and correlate with physiological processes / identification of growth factors etc.

  • Real-time delivery of antagonists and growth factors to tease out activity in biological processes.

  • Use of microfuidics and manifold to deliver growth factors to chip for long term delivery.

Assessment of Angiogensis

Assessment of tissue growth / local interactions for tumor cells

Q - How to and maintain cell metabolism ?

Vasculogenesis (Sprouting from blood vessel)

• • Place a vessel (rat femoral artery) in a hydrogel and grow the micro-vasculature out

  • Also perhaps able to re-anastamose once grown

  • Also run vessel though chip above to feed cells from bottom.

  • Encase a blood vessel (femoral artery) in a hydrogel and sprout out into that.