Development biology pathway by which load-bearing bones form was mimicked

Researchers from Indian Institute of Technology (IIT) Delhi and IIT Kanpur have used a different approach to mimic the development biology pathway by which adult load-bearing, long bones are formed. The bone construct was fabricated by combining tissue engineering and 3D bioprinting. The study also helped in understanding the detailed gene expression and sequential signaling pathways that get upregulated when embryonic-stage cartilage becomes bone-like cells.

How bones form

There are two ways in which bones are formed. In the case of cranial bones (which are not load-bearing), mesenchymal stem cells directly differentiate into bones without being converted into a cartilage. However, in the case of load-bearing, long bones, such as femur, stem cells first form a cartilage template, which then undergoes further differentiation to form bone cells. Bones formed from a cartilage template are designed to bear weight.

Till date, all attempts to develop load-bearing bones using different scaffolds have been by differentiating the stem cells directly into bone cells thus bypassing the crucial, intermediate stage of cartilage formation. “The efficacy of such bone constructs is yet to be demonstrated in bearing loads. There is very poor correlation between bone constructs developed in vitro and in vivo. Also, gene expression pattern of these tissue-engineered bones largely differ from human adult bone,” says Prof. Sourabh Ghosh from the Department of Textile Technology at IIT Delhi and one of the corresponding authors of a paper published in the journal ACS Biomaterials Science & Engineering.

In a paper published last year in the journal Bioprinting, the same team used 3D bioprinting and bioink (which contains silk proteins, mesenchymal stem cells and growth factors) to tissue engineer the cartilage.

In the latest work, the cartilage was first 3D bioprinted using bioink and cartilage characteristics were thoroughly characterised. The researchers then added a thyroid hormone (Triiodothyronine or T3) to the cartilage to facilitate the differentiation of cartilage into bone-like cells.

Stark difference

Unlike bone cells formed directly from stem cells, bones formed through cartilage differentiation in the lab exhibited a stark difference — gene and protein expressions were similar to when bone development occurs naturally in the body.

Also, the three key cellular signaling pathways for osteogenic differentiation were found to be upregulated. “When we followed a different strategy to develop bone there is more similarity to limb skeleton development in vivo,” says Prof. Ghosh.

“The load-bearing capacity of a bone depends primarily on the quality of extracellular matrix. In loading-bearing bones, the extra cellular matrix comprises 95% while bone cells are just 5%. So if you are trying to fabricate a load-bearing bone construct it is better to have more extracellular matrix,” says Prof. Amitabha Bandyopadhyay from the Department of Biological Sciences and Bioengineering at IIT Kanpur and another corresponding author of the latest paper. “Compared to bone formed directly from stem cells, the extracellular matrix of the bone construct developed through the intermediate cartilage process was 10s of times higher.”

“We followed a four-step process to develop the load-bearing bone. We first developed chondrocytes (cartilage) from stem cells and then differentiated them into hypertrophic chondrocytes. During this process, the sponge-like cartilage becomes a brittle tissue. While the brittleness is not good for cartilage, here it is following the development biology mechanism to become a bone,” says Prof. Ghosh. In the third step, the hypertrophic chondrocytes differentiate into bone-like cells (osteoblasts) and finally to adult bone cells (osteocytes).

While it takes three weeks for the cartilage to be formed from mesenchymel stem cells (chondrogenesis), it takes another two weeks for bone formation (osteogenesis).

Though the paper does not report on mechanical properties of the bone construct, Prof. Ghosh says mechanical studies carried out show better results than when the bone has been developed directly from stem cells. The team plans to undertake studies on animals.