Bone Tissue Engineering

This post is a bit of a "review of a review". It contains information that I've gleaned from an introductory paper in bone tissue engineering (TE). This is a research area I hope to pursue, so I wanted to begin at the basics. I am also learning as I go through the paper, so I will include Wikipedia links to terms that you may want further reading on. This is not exactly a summary, as I have included some of my own thoughts and I don't cover everything, but the scientifically relevant material should be attributed to the authors. The article today is:

Porter JR, Ruckh TT, Popat KC. Bone Tissue Engineering: A Review in Bone Biomimetics and Drug Delivery Strategies. Biotechnol Prog. 2009, Vol. 25, No. 6. 1539-1560. 

Problem: What's the motivation behind bone tissue engineering (or any non-specific regenerative therapy)? Generally, the goal is to restore lost or decreased function of tissue in order to regain normative or near-normative lifestyle. There are several contributors to damaged bone tissue. The most widespread disease factors include osteoporosis, osteoarthritis, and osteomyelitis (osteo- meaning "bone"). Additional factors include physical trauma, orthopedic surgeries, and aging. There is significant need for more efficient and successful therapies as the baby boomer generation reaches retirement age and the population of the elderly rise. New bone TE strategies could also greatly reduce the economic strain of repeated treatment.

Current Methods: Many diseases or injuries do not allow for healing by mechanical fixation alone (setting the bone and letting it self-repair). This results in a non-union, which is a permanent failure of healing. This can occur in traumatic bone fractures, tumor resection, or bone loss, leaving critical-size bone defects. The current method of treatment is autogenous grafting, where host bone is removed from another site (pelvis or iliac) and used to replace the defected area. Allografts from other hosts (cadavers/doners) are also possible, though these carry the risk of infection, disease transmission, or bioincompatibility (immune response leading to rejection).

Bone TE Principles: Currently, synthetic bone scaffolds are used to mimic the physiological environment of the body. More specifically, they must:

• Provide mechanical support
• Have a porous architecture for vascularization and bone growth
• Allow for bone cell migration
• Promote osteogenic differentiation (osteoinduction)
• Enhance cellular activity in scaffold/host integration (osseointegration)
• Degrade in a way that facilitates load transfer to newly created bone
• Not produce toxic byproducts
• Not incite inflammatory response
• Be capable of sterilization
• Deliver drugs in controlled manner

One of the more promising approaches involve the use of mesenchymal stem cells (MSCs). They are multipotent progenitor cells, meaning they have the ability to differentiate into several different types of tissues. For MSCs, this includes osteoblasts (bone), chondrocytes (cartilage), and adipocytes (fat). These MSCs can be seeded onto a synthetic scaffold. While on the scaffold they can be stimulated to differentiate into osteoblasts, which then produce bone extracellular matrix (ECM) ex vivo (outside the body). This scaffold can then be implanted surgically into the defect site. The scaffold then integrates with native tissue.

In this method of bone tissue engineering, there are 3 methods of ex vivo cell culturing: growth factor delivery, bioreactor systems, and gene therapy. Some growth factors that increase osteogenic activity include platelet derived growth factors, bone morphogenic proteins, insulin-like growth factors, and transforming growth factor-βs. These are added to culture media to essentially "feed" the cells. One of the biggest problems in the use of MSCs on scaffolds for implantation is the loss of phenotypic behavior of these cells when placed in vivo (inside the body). This includes osteodifferentiation and the ability to form bone, so viability is a major concern. Another problem is the need for two surgeries, one to remove and harvest MSCs from the host, and another for implantation of the scaffold. Bone also has low concentrations of MSCs and low proliferative capacity, making it difficult to achieve high density on the scaffold. 

This paper focuses on a separate approach to bone TE, involving the use of acellular scaffolds implanted at the defect site. They are infused with drugs and bioactive molecules that are released in a controlled manner as the scaffold degrades. 

• Benefits:
• Easy to sterilize
• Have a shelf life (biodegradable)
• Less prone to infection
• Challenges:
• Design of a scaffold that mimics the micro/nano-scale architecture of bone tissue
• Precise, timed, controlled release of bioactive molecules and drugs
• Match erosion of scaffold with synthesis of bone tissue

Acellular Scaffolds and Bioactive Drug Delivery: There are a few different materials that have been proposed for scaffold construction. Ceramics have been shown to be able to degrade and release drugs in a controlled manner, but have poor mechanical properties. Natural polymers are inherently biodegradable and compatible with native tissue, but they do not have tunable degradation rates (cannot adjust), have poor mechanical properties, and are difficult to sterilize. Synthetic polymers are most successful in controlled release of drugs. These molecules are either covalently bound to the polymers or embedded inside the network matrix. As the polymers degrade, the bioactive molecules are released.

Mechanism of polymer degradation: The polymers have unstable hydrolytic linkage backbones. First, random chain scission occurs, where any ester bond in the polymer has equal chance of being cleaved by hydrolysis (chemical process in which water is broken down into protons and hydroxide ions after breaking apart a polymer). When the molecular weight is reduced to a certain amount (5kDa), the polymers diffuse out of the bulk matrix. Degradation also depends on scaffold architecture, polymerization of multiple polymer types, and the presence of hydrolytic enhancers/suppressors. 

In taking into account scaffold construction and polymer selection, one must focus on: degradation rate compared to bone growth rate, non-toxic degradation products, and ease of use of polymers. I will very briefly cover some commonly used scaffold materials.

Polyesters: The most commonly used polyesters are poly(lactic-acid) (PLA), poly(glycolytic-acid) (PGA), and poly(caprolactone) (PCL). They are FDA approved and have a wide variety of use in medicine, such as in sutures, screws, stents, etc. PLA and PGA have been shown to increase osseointegration and are a good method of slow delivery of drugs. PLA has a low modulus so it must be co-polymerized with a polymer of higher modulus so it can be mechanically stable. PGA alone has a high modulus and degrades completely in 4-6 months. PLA and PCL have longer degradation rates (2+ years). PCL has a high modulus and neutral, absorbable degradation products, making it a good choice for bone TE.

Copolymers: Many researchers combine polymers to create ideal material properties through a method called copolymerization. One of the most common copolymers used is poly(lactic acid-co-glycolic acid) (PLGA). PLGA is good for delivery and encapsulation of drugs, but has poor mechanical properties, so it must be further combined with other materials. 

Growth factor and antibiotic delivery: Bone growth and healing is influenced by a variety of growth factors. However, many scaffolds have only been able to deliver single growth factors, which limit their clinical effectiveness. Research has been shown that controlled and sequential release of multiple bioactive molecules can significantly increase bone regeneration. The challenge is to achieve a highly controlled spatial and temporal delivery method. Often secondary in thought, though equally important, are the pathological factors involved in scaffold implantation. Drugs such as antibiotics, chitin, DNA, RNA, chemotherapeutics, etc, are necessary to prevent infection, or cancer recurrence (in cases of osteosarcoma). Infection can not only inhibit the regenerative process, but can also have fatal consequences. One of the glaring challenges involved with drug delivery is antibiotic viability within the scaffold. 

Challenges and Outlook: Bone TE is promising and could have extensive benefits in several arenas. However, there are also many challenges and improvements that must be made before it can be employed in clinical trials. Researcher must:

1. Create a scaffold that has sufficient mechanical properties throughout the degradative process
2. Effectively release of bioactive molecules and drugs in a highly controlled manner (spatial and temporal)
3. Direct local multipotent cells behavior towards bone regeneration

Next: Through a conglomeration of resources, I hope to address some bone anatomy in the next post.