In the present paper, the efficacy of 3D-printed PLA scaffolds both as cell-free and cell-seeded construct was assessed in the repair of a critical-sized defect in rat calvaria. Our findings suggest that both scaffolds provide efficient templates for new bone growth and repair without producing any toxic effects. The ALT and AST levels in blood serum are indicative of the systemic influence of the implanted scaffolds on liver function [
34‐
36]. In the results obtained here, the levels of these two enzymes laid below the normal levels. PLA is a synthetic polymer that is widely used in tissue engineering applications and its high biocompatibility has been reported frequently [
37‐
40]. Our findings also rejected any toxicity or abnormality caused by neither scaffold material nor the manufacturing approach.
The osteocalcin concentration was measured in blood serum. Osteocalcin is produced only by mature osteoblasts and plays a role in bone mineralization [
41]. It is mainly deposited into the bone extra cellular matrix (ECM) and only a small quantity of its newly formed reaches the circulation [
42,
43]. The highest osteocalcin level in PLA + Cell at week 8 might indicate the higher mineralization in this group (as confirmed by the result shown in Fig.
10a). The level of this osteoblastic marker at week 12 was lower in all groups and it may be due to the fact that after this time the defects were mostly filled by new bone. In one study conducted by Zhang and Zhang [
43], the osteocalcin expression by MG63 exposed to microporous chitosan scaffolds reinforced by calcium phosphate was assessed. The authors obtained lower osteocalcin concentration at day 11 compared with that of day 7. Their result on reduction of osteocalcin level by time is in agreement with our findings. Another point is that the level of this osteoblastic marker in the defect group was not changed at different time points. This is due to the fact that no bone matrix was formed at the defect site in this group.
Both PLA and PLA + Cell scaffolds showed to induce tissue regeneration at the defect site. The connective and bone tissues along with collagen fibers and blood vessels were formed around the scaffold struts. However, the histological analysis revealed that the PLA + Cell scaffolds caused better bone formation and repair. The presence of BMSCs on the scaffold appeared to help in the bone regeneration process. MSCs are known as self-renewing, multipotent cells, which exist in different body tissues and are considered as reparative cell reservoirs. These cells differentiate in response to signaling at the site of injury [
44,
45]. Furthermore, MSCs can contribute to the maintenance of stem cell niche and tissue homeostasis [
46]. Moreover, they have low immunogenicity and show effective immune-suppressive qualities. Nevertheless, the MSCs recruitment and migration from adjacent tissues to the defect site is not probably adequate for differentiation into osteogenic precursor cells in severe bone defects [
7], such as the critical-sized defect (7.6 mm) in the present study. Therefore, scaffolds can be employed to have a more effective migration of the MSCs differentiating into osteo-progenitor cells at the defect area. The promising results of our histological analysis suggest that the PLA scaffold provided an appropriate environment for the viability of the BMSCs. This can be attributed to the scaffold structural characteristics including proper biomaterial composition, porosity percentage, and pore sizes [
8,
47‐
49]. These properties along with mechanical stability, stiffness, biodegradation, and non-toxicity are required for the successful performance of an implanted scaffold [
50,
51]. PLA has this combination of properties which results in an acceptable function in-vivo. The only drawback of PLA is its hydrophobicity and reduced cell adhesion [
52]. Therefore, some studies focused on PLA modification using bioceramics or surface treatments [
53,
54]. It would be interesting to study the in-vivo performance of the treated PLA and its composites along with BMSCs to find out about the synergistic and antagonistic effects. Another issue that can be considered in future studies is the use of growth factors that can provide the proper signaling and help in stem cell differentiation [
55].