Autologous bone, inert alloplastic materials, or combinations thereof are today used in cranial reconstructions. Materials used in conventional alloplastic cranial implants are plastics, such as polyether ether ketone, poly(methyl methacrylate), and polyethylene, and metals such as titanium. Although these materials are considered biocompatible, there are major risks for long-term complications, such as extrusion through the skin and infection.1–3 Recent publications report complications requiring surgical intervention in 20–30% of the cases treated with these methods.1–9 In this context, the development of alternative techniques appears essential, for example, bone regenerative materials. Osteoconductive implants are based on materials that allow ingrowth from adjacent host bone. Although favorable, osteoconductive properties may not be sufficient to heal large bone defects. Osteoinduction, the stimulation of resident or circulating mesenchymal stem cells to differentiate into osteoblasts, may be required to potentially heal segmental bone defects.10 However, neither osteoinductive cytokines, such as bone morphogenetic protein-2, nor certain bioceramics, with proposed combined conductive and inductive properties, have successfully been used for large bone defect repair in a clinical setting.11–15 We recently reported the development of a bioactive calcium phosphate–based cranial implant (OssDsign, Uppsala, Sweden) used in a therapy-resistant patient.16 Bone growth was indicated by 18F-fluoride positron emission tomography/computed tomography after 27 months. The aim of the present investigation was to show new bone formation induced by the ceramic implant. We report concluding results from 2 patients from whom the bioactive implants were either replaced due to aesthetical concerns or surgically exposed for the removal of fixating titanium plates, respectively. This gave an opportunity to inspect the reconstructed areas and obtain biopsies 9 and 50 months after surgery for gene expression analyses and histological examinations.
MATERIALS AND METHODS
Mosaic-designed calcium phosphate implants were manufactured with molding technique as described previously.16 Both patients were given written information about the procedures. Informed consent was obtained with signed approval to take perioperative biopsies.
Patient 1 was a 41-year-old man who suffered from previously infected bone flap and failed polymethyl methacrylate implant after neurosurgical intervention for the treatment of chronic infection in the frontal sinus area. The frontal bone defect measured approximately 60 cm2. A customized calcium phosphate implant was manufactured and implanted. Because of aesthetical concerns with a flat contour of the forehead, the implant was surgically removed, and the ceramic implant was replaced after 9 months. Tissue samples for histology (n = 3) and gene expression (n = 10) were obtained. RNA was extracted and reversed transcribed according to manufacturer’s instructions using TATAA GrandScriptTM kit (TATAA Biocenter, Gothenburg, Sweden). Samples were amplified on the LightCycler 480 System (Roche Applied Science, Germany). The genes of interest coded for osteopontin, osteocalcin, collagen 1, calcitonin receptor, and cathepsin K. Biopsies for histological analyses were fixated in formalin, decalcified in formic acid solution, and dehydrated before embedded in paraffin. Ten-micrometer sections were stained with hematoxylin eosin.
Patient 2 was 33 years old and had an approximately 35 cm2 parietal defect after trauma. He was primarily reconstructed with the ceramic implant 50 months earlier. Lately, the patient had complaints with local discomfort from fixating titanium plates, and indication for reentry was the removal of the plates. Tissue sample from a ceramic tile located at the central part of the implant was obtained by the use of bone nipper. Histological sections were prepared as described above.
Patient 1 (9 Months Postoperatively)
The implant was inspected and appeared without macroscopic evidence of bone deposition (Fig. 1). Histology revealed collagen fibers and blood vessels, whereas no bone was detected (data not shown). Occasional multinuclear cells were detected. In the border between the calvarial defect and the preexisting parietal bone, newly formed bone was in direct contact with the surface of the ceramic tiles.
Gene expression analysis demonstrated a markedly higher expression of osteopontin, osteocalcin, and collagen 1 in the central defect and border sites compared with the parietal bone and soft tissues (Fig. 2). The calcitonin receptor and cathepsin K showed higher expression in the bone defect and in the border to parietal bone than in the parietal bone itself and soft tissue, irrespective of location (data not shown). The expression of transcription factor runx2 was low in all samples tested.
Patient 2 (50 Months Postoperatively)
Gross inspection showed bleeding bone that appeared to cover all ceramic tiles (Fig. 3A). Tiles located heterotopically at the border of the implant seemed integrated with host bone, whereas solid ectopic bone growth appeared on ceramic tiles located in the middle of the implant without bone bridging between tiles. Histological examination from this area revealed compact bone in direct contact with remnants of inert ceramic materials (Fig. 3B). Blood vessels were present within the compact bone.
Although caution is a prime requirement when concluding biological processes based on analyses in few patients, the present case studies provide important information with respect to the feasibility of regenerating bone in major cranial defects. Implants with osteoconductive and possibly osteoinductive properties were shown to induce bone healing of cranial defects in patients as demonstrated by gene expression analyses and histology. The ceramic compound of the implant, comprising monetite, β-calcium pyrophosphate (PPi), β-tricalcium phosphate, and brushite, is intended to chemically resemble native bone and to be part of the process of coupled bone formation. Gene expression analysis 9 months postoperatively indicated osteoclastic activity in parallel with new bone formation. This suggests that cell-mediated resorption of calcium phosphates occurs, which may be a prerequisite for deposition of new bone.17,18 However, the process implicates a narrow balance between resorption rate and new bone deposition. PPi is essential because removal of this phase provokes enhanced resorption and complete resolution of ceramics within months without new bone formation as shown in large animals (not published). Thus, PPi plays an essential role in controlling resorption rate of calcium phosphates and differentiation of osteoprogenitors and, as a consequence, new bone formation.19,20 A chart review including more than 100 patients is currently underway to assess clinical outcome and complication rates in patients reconstructed with the use of the bone-stimulatory cranial implant.
A bioactive calcium phosphate-based implant was developed as an alternative to conventional inert alloplastic materials used for cranial repair. The implant was shown to stimulate bony healing of cranial defects as demonstrated by gene expression analysis and histology in two patients. We hypothesize that the bone regenerative effects may be advantageous by reducing complication rates in cranioplasty procedures as compared to the use of plastics and titanium.
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