Evaluation Of The Osteogenesis Of 3D Printing PLGA/HA Scaffolds In A Rat Femur Model And Its Biocompatibility

Objective: We analyzed the biocompatibility of biodegradable poly(lactic-co-glycolic acid)/ hydroxyapatite(PLGA/HA)bioactive scaffolds prepared by 3D printing technique,the repair effect of defects and histopathological changes during bone healing with the application in the full-thickness femoral bone defect in rats.Methods: 1.We have rapid-prototyped the 3D printing poly(lactic-co-glycolic acid)-hydroxyapatite(PLGA/HA)bioactive scaffolds to evaluate the effect and biocompatibility of in repairing rat full-thickness bone defect in vivo and in vitro.2.In vitro: The MC3T3-E1 osteoblast cells were seeded on the 3 mm × 3 mm × 2 mm PLGA/HA aseptic scaffolds.Following fixation in 2.5% glutaraldehyde,a gradient concentration of ethanol solution with step-by-step dehydration,critical point drying and gold spraying,the samples were placed in SEM to take the photographs,which was performed to further investigate the cell morphology and distribution in the scaffolds at day 14 of culture.3.In vivo: Thirty female Sprague Dawley(SD)rats aged 6 weeks were intraperitoneally anaesthetized with chloral hydrate,and a 2-cm-long incision was created on the skin along the long axis of the femur of both hind legs.The fascia,muscle and periosteum were bluntly separated in turn.A slow-speed drill was used to grind out a4-mm-long full-thickness bone defect that was deep into medullary cavity nearly 1/3 of the length of each femur on the proximal aspect.A PLGA/HA scaffold was implanted into the left defect(Fig.1B)with no implant placed in the right side.The bilateral femurs containing 1.5 cm of normal bone tissue from rats at various developmental stages(3days;1,2,3,4 and 5 weeks)were collected postoperatively.Following decalcification,dehydration,translucency and paraffin embedding,those specimens were processed by routine histological procedures to analyze the histopathological changes of bone wound healing.In addition,the steoblast-related genes of IGF-1,COL-1,OC and OPN were detected by real-time polymerase chain reaction(RT-PCR)postoperatively to further evaluate the bone repairing distinctions between the two groups.Results: 1.The PLGA/HA scaffold prepared by 3D bioprinting technology was characterized by a three-dimensional cubic structure with homogeneous and interconnected high-density pores in the interior.2.Under SEM,our bioactive scaffold featured well-distributed and interconnected porous latticework,a place that the mouse osteoblasts penetrated into and their processes were closely attached to the material,which presented good growth conditions.3.Gross morphology of bilateral femur specimens showed that the repairing process of bone defect in experimental group is faster than that in control group.By the third week,the reconstructive bone mass had progressively grown into a defect as the scaffold's degradation evolved,and the edge was consistent with that of the normal bone.The neo-formed bone resembled normal bone in terms of color,shape and tissue quality by fifth week.In contrast,hyperemia of the regenerating tissue within the control defect began to decline,but there was still a significant difference in the morphology compared to that of original bone.4.Finally,HE staining images demonstrated that the cortex of the new bone in the experimental femur reproduced the structure of the surrounding normal bone tissue combined with complete scaffold degradation.The control groups,on the other hand,suffered a long duration of healing with a poor osteogenic effect.5.The expression level of osteoblast-related genes in the test limbs were up-regulated higher than control group.Conclusion: 1.Taken together,our innovation in this work demonstrated that the 3D printing PLGA/HA bioactive scaffolds had excellent biocompatibility and osteogenesis.Cell experiments suggest that the PLGA/HA scaffold not only provided a track for cell migration,but it also promoted the adhesion of osteoblasts on the material.2.Animal experiments show the PLGA /HA scaffold's ability to accelerate the formation and reconstruction of new bone,adapt well to the movement of the load-bearing part of femur,and promote the repair of bone defect in rats as well as the excellent biodegradability,which provides a powerful theoretical basis for PLGA/HA biomaterial to be potential candidates for bone grafting for future orthopedic applications.