Research Proposal Page

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Mahesh Vidula and Michael Meyer 20.109 TR Red

Proposal Topic: Bone Tissue Engineering

Background of problem: A current bone tissue engineering strategy consists of growing mesenchymal stem cells on scaffolds, inducing them to differentiate into osteoblasts, and culturing these cells on the scaffold. Then, the osteoblasts will begin to deposit bone matrix, in a process known as intramembranous ossification. However, this protocol is not very successful in in vivo experiments.

Therefore, researchers have begun looking into endochondral ossification, which involves the formation of cartilage. After cartilage forms, osteoblasts penetrate the cartilage matrix and begin to deposit bone matrix.

However, endochondral ossification usually take up to 3-4 weeks, which is a long time. Therefore, we aim to accelerate the process of in vitro bone formation using ultrasound stimulation.

Project Idea: It is interesting and realistic to conduct a project that involves differentiating mesenchymal stem cells on a compatible scaffold to chondrocytes, and then differentiating these chondrocytes to hypertrophic chondrocytes. Both of these differentiations can be accelerated with ultrasound. The hypertrophic chondrocytes will then form a cartilage matrix. Osteoblasts can be cultured on this matrix, and they can be used to grow bone tissue (also can be accelerated by ultrasound) in vitro.

Also, another possibility is to take the cartilage matrix (produced by hypertrophic chondrocytes), and implant it in vivo. This has been shown to lead to bone formation, due to either osteoblasts in the matrix, or growth factors secreted by hypertrophic chondrocytes (Jukes et al. 2008).

Overall: Ultimately, we will provide a method to accelerate the formation of bone tissue directly from MSCs, a process that usually takes much longer.

Summary - The enhancement of bone regeneration by ultrasound, Lutz Claes, Bettina Willie: Bone fractures and bone-related diseases are extremely common (6.2 million fractures/year in the US alone). While many of fractures heal by themselves, 5-10% will exhibit delayed healing and some will never heal.

Low intensity pulsed ultrasound (LIPUS) has been shown to increase the regenerative capacity of bone in vitro. The mechanism is unknown, but it is possibly related to the slight heating of some enzymes shown to respond significantly to these small fluctuations in temperature. Acoustic streaming and cavitation may affect membrane permeability and diffusion and could also lead to increased bone formation. Micromotion has been hypothesized to provide mechanical stimulation.

Results gathered so far show a high dependency of stimulation on the frequency of the ultrasound; a carrier frequency of 1.5 MHz has been shown to stimulate osteogenesis experimentally and clinically.

During secondary fracture healing, bone forms via two mechanisms: intramembranous bone formation and endochondral ossification. Endochondral ossification (calcification of cartilage and replacement by bone) is shown to be influenced by LIPUS while this method is shown to have no effect on intramembranous bone formation.

On individual cells (mesenechymal stem cells, fibroblasts, osteoblasts, and chondrocytes) in vitro studies of ultrasound show an associated increase in protein synthesis and collagen synthesis. Studies of the effect often quantify the bone markers osteocalcin and alkaline phosphatase. A specific study in vitro shows that LIPUS stimulated the expression of c-fos and cyclooxygenase-2 genes and elevated mRNA levels for the bone matrix proteins alkaline phosphatase and osteocalcin.

Tissue culture studies showed a stimulation of osteoblast activity as well as hypertrophic cartilage cells as a result of LIPUS.

Overall in vivo studies have shown that LIPUS treatment does not stimulate osteogenesis in intact bone and does not affect bone remodeling. The real effects of LIPUS are observed in vitro and during the soft callus formation phase.

Summary - Endochondral bone tissue engineering using embryonic stem cells Jukes JM, Both SK, Leusink A, Sterk LMT, van Blitterswijk CA, de Boer J. PNAS 2008; 105:6840-5.

The authors strove to grow bone tissue using endochondral ossification. Realizing that differentiating embryonic stem cells (ESCs) to osteoblasts did not give successful in vivo results, the researchers opted for a different technique. They first seeded mouse embryonic stem cells on a scaffold, and differentiated these cells to chondrocytes. The chondrocytes formed cartilage, and this cartilage construct was implanted into immunodeficient mice. Three weeks later, bone tissue formed.