脊椎骨欠損部における脂肪由来幹細胞の骨形成促進効果

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  • Enhancement of bone formation with adipose-derived regenerative cells in large spinal defects
  • セキツイ ホネ ケッソンブ ニ オケル シボウ ユライ カンサイボウ ノ コツ ケイセイ ソクシン コウカ

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脊椎腫瘍に対する腫瘍脊椎骨全摘術 (total en bloc spondylectomy, TES) のような巨大な骨欠損を生じる手術では，より早く確実に骨癒合する椎体再建方法が求められる．本研究では，動物実験モデルにおいて脂肪由来幹細胞 (Adipose derived regenerative cells: ADRCs) を椎体再建材料として用い，移植骨の骨形成への関わりを骨形態学的に評価した．犬の第13胸椎TESモデル12例を用い，ADRCs群と対照群に分けた．対照群は自家骨を， ADRCs群は自家骨と犬の背部脂肪組織から抽出したADRCsを用いて椎体を再建した．各群術後2週と4週に検体を回収し，ヴィラネバ骨染色した後，正中矢状断の非脱灰研磨切片を作製した．光学偏光顕微鏡で骨形態計測を行い比較検討した．ケージに占める線維性結合組織の割合は，2週対照群27.5%／ADRCs群65.5%，4週対照群58.3%／ADRCs群86.6%，新生骨に占める層板骨の割合は，2週対照群7.9%／ADRCs群47.5%，4週対照群63.3%／ADRCs群96.4%であった．骨形成速度 (mm3／mm2／day) は，2週対照群0.06 ／ADRCs群0.31，4週対照群0.66 ／ADRCs群1.95であった．脂肪由来幹細胞を用いると線維性結合組織の増生に始まる移植骨の一連のリモデリング量が増加し，骨形成が促進されると考えられた．

After total en bloc spondylectomy (TES) for complete resection of malignant spinal tumors, spinal reconstruction is performed for critical-sized bone defects using a titanium cage filled with bone graft. Reconstruction materials that promote bone healing are needed for strong initial fixation and biological bone healing. Meanwhile, regenerative medicine has evolved recently. Adipose-derived regenerative cells (ADRCs) are easily isolated in large amounts from autologous adipose tissue, safe to use, and simple to apply clinically. We examined whether ADRCs promote osteogenesis of bone graft inside a cage in a canine TES model. We used 12 female dogs with (average weight, 10.5 kg) aged 12 months. The dogs were divided into ADRC (n = 6) and control groups (n = 6). In the ADRC group, about 10 g of adipose tissue was harvested from the neck preoperatively. ADRCs were isolated using a previously reported method. ADRCs and an autograft with fibrin glue were packed into a titanium cage. In the control group, only the autograft with fibrin glue was packed into the cage. TES was performed in the 13th thoracic vertebra. For spinal fixation, pedicle screws were inserted (2 each) above and below the thoracic vertebra . The dogs were given calcein to label the mineralizing bone 9 and 2 days before killing them, and were killed at 2 weeks (3 dogs from each group) or 4 weeks after surgery (3 dogs from each group). Specimens were harvested, fixed in 70% ethanol, stained with Villanueva bone stain, and ground to thickness <20 μm through the cage’s long axis. The bones were histomorphologically assessed by using fluorescence polarization microscopy. In all cases, the connective tissue expanded inside the cage from both ends toward the center. The ratio of the connective tissue volume to the total volume (CV/TV) at 2 and 4 weeks was higher in the ADRC group (control group: 27.5% and 65.5%, respectively; ADRC group: 58.3% and 86.6%, respectively). More blood vessels in the connective tissue were observed at 2 and 4 weeks in the ADRC group (ratio of the total length of blood vessels to field area: control group, 0.00294 and 0.00347, respectively; ADRC group: 0.00483 and 0.00680, respectively). The localized area adjacent to the vertebra at either end of the cage had bone formation. Lamellar bone was formed in the ADRC group, whereas almost all woven bone was formed in the control group 2 weeks postoperatively . The ratio of lamellar bone volume to new bone volume (LBV/NBV) increased in both groups 4 weeks after surgery (control group: from 7.9% to 47.5%, ADRC group: from 63.2% to 96.4%). Under fluorescent light, few depositions of calcein were observed in the control group, while diffuse deposition, which showed immature lamellar structure, was observed in the ADRC group 2 weeks after surgery. The orderly labeled line indicates the mature lamellar structure in both groups 4 weeks after surgery. The bone formation rate (BFR), calculated by using the parameter from the fluorescence labeled line, increased significantly in the ADRC group 4 weeks after surgery (control group: from 0.06 to 0.66, ADRC group: from 0.31 to 1.95). In general, bone healing of the autograft follows 4 processes: (1) vascular rich connective tissue extension, (2) resorption, (3) new bone formation, and (4) mineralization. Our results suggest ADRCs confer advantages in processes (1), (2), (3), and (4). Therefore, the use of ADRCs as materials of spinal reconstruction promotes early-stage bone formation of the autograft inside a cage.

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