In Vitro and In Vivo Testing of Biomaterials and Cell/Surface Interactions

In Vitro and In Vivo Testing of Biomaterials and Cell/Surface Interactions

Introduction

  • Speaker: John L. Ricci, PhD, FBSE

  • Department: Molecular Pathobiology, Division of Biomaterials and Regenerative Biology, NYU College of Dentistry

  • Overview of topics:

    • Cell-Free In Vitro Testing of Biomaterials

    • Cell-Based In Vitro Testing of Biomaterials

    • Animal Screening and Application Models

    • Biomaterials Research

    • Materials Testing

    • In Vitro testing

    • In Vivo Testing

    • Clinical Testing

    • Characterization: mechanical, bulk chemistry, surface chemistry, degradation

    • Basic tripartite testing: cytotoxicity, carcinogenicity, mutagenicity

    • Cell-surface response and tissue interaction

    • Human clinical trials for application testing

Cell-Free In Vitro Testing of Biomaterials

Characteristics of In Vitro Testing

  • Involves observing degradation/changes in biomaterials in cultures or simulated body fluids (SBF).

  • Focus: Understanding the interaction between biomaterials and biological components without the influence of live cells.

Calcium Sulfate as Bone Graft Material

  • Observation: Mineral deposits in tissue adjacent to Calcium Sulfate (CS) implants.

  • Questions addressed:

    • How does Calcium Sulfate (CS) work?

    • What is the speed of resorption?

    • How does it affect bone healing?

    • Reaction with body fluids?

In Vitro Study with CS

  • Techniques employed: Backend Electron Imaging (BEI) and X-ray Microprobe (XRM)

  • Analysis of CS samples in water and simulated body fluid (at 37°C):

    • Findings:

    • Mass loss in SBF was significantly higher than in H2O.

    • Notes flaking of CS surface in SBF.

    • Hypothetical query: Is this a precipitation reaction and does it speed dissolution?

Collagen Onlay Study

  • Added SBF at 37°C to evaluate reaction at interface with collagen.

  • Findings indicate mineral deposits observed are not CS, but calcium phosphate precipitate resulting from CS interaction with body fluids.

Other Examples of Cell-Free Studies

  • Drug/growth factor release studies from biomaterials.

  • Mass loss studies of controlled solubility materials, assessing mechanical strength loss and changes in molecular weight of resorbable materials, such as sutures.

Cell-Based In Vitro Testing of Biomaterials

Purpose

  • Why execute Cell-Based In Vitro Testing?

  • Focuses on tripartite testing: cytotoxicity, carcinogenicity, and mutagenicity (ASTM G87-1).

  • Aims to simulate the biomaterial/tissue interface, analyzing interactions closely.

Cell Interaction Parameters

  • Selection of the biomaterial surface as cell culture substrate.

  • Appropriate cell types:

    • Primary cells from tissues, e.g., rat fibroblasts, osteoblasts.

    • Transformed/immortalized cell lines, e.g., MG-63 osteosarcoma cells, MC-3T3 mouse cells with various phenotypes.

  • Parameters to study include:

    • Attachment, migration, growth, and differentiation characteristics.

  • **Differentiation Parameters: **

    • Alkaline phosphatase expression, osteopontin, osteocalcin, bone sialoprotein, BMP, and gene expression.

Pseudotissue Cultures and Migration/Growth Studies

  • Focus on organoids, “dot cultures” involving cells in collagen, and strategies with fibroblasts and osteoblasts.

In Vitro Microtexture Studies

Introduction to Microtexture

  • Textured surfaces were found to inhibit fibroblast colonization, affecting tissue interaction.

  • Study shows fibroblast colonies on both flat and textured surfaces, emphasizing responses to surface morphology.

Specific Studies and Outcomes

  • Authors: Kunzler TP, Drobek T, Schuler M, Spencer ND.

  • Published: Biomaterials 28 (2007) 2175–2182.

  • Findings:

    • Microgrooved surfaces produced by silicon wafer templates led to inhibited fibroblast colonization and directional colony formation (Contact Guidance).

    • Optimal groove characteristics for inhibition of fibroblast colonization are 6–12 μm width with 12–24 μm repeat spacing and heights >2 μm.

Summary of In Vitro Cell/Biomaterial Interactions

  • Various cell-free and cell-based in vitro models exist, each showcasing fundamental properties of biomaterials.

  • Limitations:

    • Models do not replicate inflammatory or immune responses.

    • Simulations represent only a small fraction of tissue response.

In Vivo Studies

Rationale for In Vivo Research

  • Necessary to conduct live animal studies post in vitro studies to measure immune responses and interactions with multiple cell types and tissues.

    • Limitations of in vitro models are acknowledged, as they cannot mimic the complete biological environment.

Advantages and Disadvantages of In Vivo Studies

  • Advantages: Understanding of complex cell/tissue response and device performance under actual clinical conditions.

  • Disadvantages:

    • Animal responses often do not represent human conditions accurately

    • Animal studies are costly, time-consuming, and ethically sensitive.

Typical Animal Study Example

  • 15 rabbits in transcutaneous implant study with four implants per animal (totaling 60 implants).

  • Includes 30 control and 30 experimental group, with various evaluation methods (light microscopy, histology, electron microscopy) costing around $23,000.

Ethics and Regulations in Animal Research

IACUC Guidelines

  • All research involving animals must obtain approval by the Institutional Animal Care and Use Committee (IACUC) which is overseen by the US Department of Agriculture.

  • Must justify species choice, infliction of pain, and alternatives considered.

Animal Research Costs

  • Per diem, surgical care, necropsy, and processing costs all contribute to the financial burden.

  • Costs across different species vary (e.g., mouse, rat, rabbit, dog, etc.), with per diem costs noted as follows:

    • Mice: $.34–.81 per day

    • Rabbits: ~$160.00–245.00 based on availability.

Types of Tests in Animal Models

Screening and Application Tests

  • Screening tests provide basic information, whereas application tests focus on specific uses of materials.

  • Evaluation methods include:

    • MicroCT, histology, histomorphometry, and mechanical testing.

Recommended Animal Models

  • Examples:

    • Controlled surgical defect models, implantable chamber models, intraoral wound healing models.

  • Evaluation using specialized methods (e.g., Mechanical testing, biochemical analysis).

Conclusions

  1. In vitro models are critical for basic biomaterial assessment but have significant limitations.

  2. In vivo studies, though complicated, provide a more complete biological context that cannot be achieved through in vitro alone.

  3. Scrutiny regarding the ethical implications and financial aspects of animal research should guide the design and justification of such studies.

Questions and Controversies

Paradigms in In Vitro and In Vivo Research

  • Skeptical views raised on the necessity and justification of using higher animal models such as primates when alternatives are available.

  • Questions of experimental design and relevance are brought forward, emphasizing ongoing debates in biomaterials research ethics and efficacy.