Imagine a small hole in the tibia of a laboratory rat, three millimeters wide, deliberately created using a low-speed dental drill. The hole was then filled with a white material derived from crustacean shells and synthetic bone minerals. Three weeks later, under a light microscope, new bone had grown to fill the gap and was more mature than that observed in the group allowed to heal spontaneously.
This is the essence of the findings published by Anne Handrini Dewi and Dr. drg. Andi Triawan, Sp.Ort. in The Indonesian Journal of Dental Research (2011). The study examined a combination of carbonate apatite (C-Ap) and chitosan as a bone substitute material, a promising approach to a clinical problem that has yet to be ideally resolved: large bone defects.
When Bone Cannot Heal on Its Own
Millions of people experience bone damage every year as a result of trauma, tumors, or degenerative diseases. The best option to date has been autogenous bone grafting, in which bone is taken from the patient's own body. Although effective, the procedure leaves a wound at the donor site. Synthetic alternatives have also continued to evolve, ranging from natural coral and demineralized bone matrix to bioactive ceramics and various types of calcium phosphate.
Among these candidates, carbonate apatite has attracted attention for one fundamental reason: it is the mineral that actually constitutes human bones and teeth, rather than pure hydroxyapatite as is often assumed. Meanwhile, chitosan, a natural polymer produced through the partial deacetylation of chitin from insect and crustacean shells, is biocompatible, biodegradable, antibacterial, and capable of accelerating wound healing.
Combining the two is a logical hypothesis. The question is: does this combination actually work inside a living organism?
A Hole in the Tibia, an Answer Under the Microscope
The researchers used 18 three-month-old male Sprague Dawley rats weighing 250–300 grams. Defects measuring 3 × 1.25 × 1 mm³ were created in the left and right tibiae of each rat using a low-speed dental drill with continuous saline cooling. The defects were treated with three different interventions: C-Ap alone, a C-Ap–chitosan combination at a 1:1 ratio, and a control group without any implanted material.
The rats were sacrificed during the first, second, and third weeks. The bone specimens were then processed histologically, stained with Hematoxylin and Eosin, and examined under a light microscope.
The results were reflected in the numbers. Two-way ANOVA showed a significant difference in the number of osteoblasts among the groups (p < 0.05). During the first week, both treatment groups showed higher numbers of osteoblasts and osteoclasts than the control group, indicating more active bone formation. During the second and third weeks, however, the pattern was reversed: the number of osteoblasts in the treatment groups decreased, while it remained high in the control group.
"The decrease in the number of osteoblasts during the third week indicates an increase in the calcification process of osteoblasts into osteocytes,” the researchers wrote in their discussion.
In other words, bone formation in the treatment groups was not slowing down. Rather, it had progressed further—from the formation phase to the maturation phase. While the control group was still struggling to close the defect, the treatment groups had already approached the remodeling stage.
Slowly Dissolving Chitosan, Fast-Acting C-Ap
One interesting finding emerged from the histological observations during the first and second weeks: residual chitosan was still detected in the defect area. This indicated that chitosan was absorbed by the body more slowly than carbonate apatite. Consequently, C-Ap alone was shown to be more effective in increasing the numbers of osteoblasts and osteoclasts during the first week after implantation.
However, the C-Ap–chitosan combination was not without its advantages. Bone matrix deposition occurred more rapidly in this group, and the interface between the old and newly formed bone showed good integration. Chitosan, with its flexible properties and ability to be formed into a paste, powder, or film, also facilitates clinical application at defect sites with irregular shapes.
The researchers concluded that C-Ap–chitosan is a potential candidate for use as a bioresorbable bone substitute. They also recommended further modifications, including converting chitosan powder into nanoparticles to optimize its absorption in bodily fluids.
From shrimp shells in the LIPI Yogyakarta laboratory to the tibiae of Sprague Dawley rats at LPPT UGM, the journey of a biomaterial toward clinical application remains a long one. But the three-week data are already sufficient to show that new bone can grow—and that humans may one day no longer need to borrow bone from another part of their own bodies.
Authors: Nanda Ayu, drg. Achmad Zam Zam Aghasy, M.Kes.
Photo: Pexels