Free periosteal graft formation of bone or cartilage has been demonstrated morphologically and biochemically, but is limited to a relatively small area. Clinical cases of near-ankylosis or already fibrous ankylosis of the hip joint in patients with ankylosing spondylitis aged 18 to 30 years are more common, and restoring hip function in such patients and avoiding premature artificial joint replacement is a major challenge in clinical treatment. In this regard, we conducted an animal experimental study of large autologous free periosteal grafts in order to further guide the clinical development of reconstruction of hip function.
1.Materials and methods
(1) Experimental animals and groups Sixteen healthy mongrel dogs, 10-12 kg each, male and female, were randomly divided into two groups: 12 in the experimental group and 4 in the control group, with the right side selected as the operated side.
(2) Surgical method: The experimental animals were anesthetized with thiopental sodium (35 mg/kg) intraperitoneally, and under aseptic conditions, an anterolateral incision was made at the hip joint, the skin was cut along the iliac crest, the subcutaneous tissue was separated, and the periosteum was sharply separated outside the periosteum with ophthalmic scissors to a range of 3 cm×3.5 cm, and the periosteum was sharply separated with a bone knife at an angle of 45 toward the iliac surface for backup, the hip joint was exposed, the femoral head was dislodged, and the femoral head was bitten off. The proximal 2/3 of the articular cartilage, deep to the bleeding cancellous bone, was contoured and rounded, and the periosteum was adhered to the trimmed femoral head with a frozen fibrin adhesive (from Shanghai Institute of Biological Products) melted in the adjacent time, with the germ layer facing the joint cavity. To prevent avulsion, 4 to 6 stitches were fixed to the peripheral cartilage with 3-0 noninvasive sutures, flushed with gentamicin solution, returned to the femoral head, and sutured layer by layer. In the control group, only the articular cartilage was removed and no periosteal graft was made. Postoperatively, gentamicin was injected intramuscularly two times, 160,000 U each time.
(3) Observation method: After surgery, the hip joint movement and incision healing of the experimental animals were observed, and the animals were executed in batches at 2, 4, 8 and 12 weeks postoperatively, with 3 animals in each batch and 1 in the control group. double layer staining JEM-1200 transmission electron microscopy observation.
2.Results
(1) General condition of experimental animals: Within 1 week after surgery, the experimental animals had limited activity on the surgical side and were in a self-protective state with a jumping gait. After 2 weeks, the animals were able to move freely and the incision was healed at stage I.
(2) Visual observation: 2 weeks after surgery, there was no slippage of the grafted periosteum, but there were folds formed, the periosteal graft site was white, soft fibrous tissue filled, the surface was not smooth, and the grafted periosteum could still be torn off. At 4 weeks postoperatively, the periosteal graft site was white with tough cartilage-like tissue, and some parts were higher than normal cartilage. At 8-12 weeks after surgery, the graft area was similar in color to normal cartilage, hard and smooth, with good curvature of the femoral head. In the control group, the defective area was filled with white soft fibrous tissue and the surface was not smooth.
(3) Morphological observation: The femoral head of the control animals showed dense collagen fiber bundles with irregular arrangement and flattened nuclei between the bundles under light microscopy. The bone surface was dense, and the outer layer was loose and clearly demarcated. The cells were shuttle-shaped with protrusions and had a small amount of rough endoplasmic reticulum and nucleoprotein bodies in the cytoplasm under electron microscopy (Figure 1, 2).
Toluidine blue staining showed that the proliferating periosteum was blue in color and the fibers between the periosteum and the trabecular bone of the recipient area became lax.
At the fourth week (28 days) after surgery, the cells of the grafted periosteum were seen to change from flat to round or oval and dense on the trabecular surface of the bone near the recipient area under light microscopy. On electron microscopy, the cells were irregularly shaped, with a large number of rough endoplasmic reticulum, polymorphic nucleoproteins and free nucleoprotein bodies in the cytoplasm, and some cells had two nucleoli in the nucleus (Figure 4, 5).
At the 8th postoperative week (56 days), hyaline cartilage was formed, and light microscopy showed small chondrocytes in the superficial layer and homologous cell populations in the deeper layer. On electron microscopy, the cartilage was irregularly shaped with a large number of microvilli. A large number of expanded rough endoplasmic reticulum was seen in the cytoplasm (Figure 6, 7).
At the 12th postoperative week (84 days), the morphological changes under light and electron microscopy were the same as at the 8th week.
3. Discussion
Animal experiments and clinical applications of smaller free periosteal grafts have been reported, but studies of large free periosteal grafts are uncommon. The size of the area should be a relative concept, depending on the spatial geometry of the articular surface, and only wounds where the corresponding articular surface can directly squeeze the biograft and affect its survival are considered large. Large cartilage defects are mostly seen clinically and have surgical value, while large periosteal grafts with a large contact area corresponding to the articular surface are prone to necrosis due to direct compression.
Therefore, we preserve the peripheral articular cartilage portion in animal experiments to reduce the compressive stress on the grafted periosteum. In addition, the dog’s hip joint moves mainly in the anterior-posterior direction, and the head-molar relationship fits relatively closely. The contact between the peripheral portion of cartilage and acetabular cartilage greatly reduces the direct pressure on the proximal 2/3, and the unilateral surgery, the protective response of the animals, and the early non-weight bearing, significantly reduces the pressure on the grafted periosteum, and this measure has an important role in the survival of the grafted periosteum and its transformation to chondrocytes.
Histological results show that the evolution of large periosteal free grafts is consistent with that of smaller periosteal free grafts. Successful clinical attempts at large-area periosteal free grafts can be expected with the application of CPM devices with traction-regulated joint pressure.
Several issues should be noted for autologous periosteal free grafts.
(1) selection of periosteum Peng Jisheng et al. found that iliac periosteum is superior to other parts of periosteum in terms of thickness of the germinal layer, cell count and vascular distribution. It is also easy to cut and has a large donor area, especially for the reconstruction of femoral cartilage, and a larger area of periosteum can be cut within the same incision, reducing patient pain. Therefore, iliac periosteum is a more ideal tissue source.
(2) Fixation of periosteum The free periosteum can only be viable if it is in close contact with the bone with blood supply. In our experiment, we used fibrin adhesive plus non-invasive nylon suture fixation method for early and exact fixation, which effectively prevented the slippage of the periosteum.
(3) Periosteal excision The greater the number of cells in the periosteal germinal layer, the higher the chances of repair transformation. Therefore, the periosteum should be excised to maintain the integrity of the periosteum to the maximum extent possible without damaging or slightly damaging the cells of the periosteal germinal layer. When we cut the iliac membrane, we use a sharp bone knife with a 45 angle toward the outer plate of the iliac bone, the force is mainly on the outer plate of the iliac bone, and the cells of the periosteal growth layer are almost uninjured, which provides a guarantee for the regeneration and transformation of the periosteal graft.