Injectable Bone Substitute Based on β-TCP Combined With a Hyaluronan-Containing Hydrogel Contributes to Regeneration of a Critical Bone Size Defect Towards Restitutio ad Integrum

Radiological evaluation of the healing processes of the femoral bone defects treated with the injectable bone substitute (IBS) (a–c) and the control (d and f). (a) The radiographs show the implantation area of the IBS (red dashed line) 1 month after treatment. Within the implantation area, the principal observation was a bulk-like radiopaque mass (red asterisks) that appeared to represent the IBS. Within the peripheral defect regions, a trabecular bone tissue ingrowth (green asterisks) was seen at this time point, whereas the center of the implantation area seemed to be free of newly formed bone. (b) Three months after treatment, further regeneration of the trabecular bone structure (green asterisks) was observed within the peripheral regions of the defect area (red dashed line), whereas in the region of the implanted IBS a radiopaque center (red asterisks) was present, indicating massive bone ingrowth. (c) Six months after implantation, a normal trabecular bone structure (green asterisks) within the former defect area (red dashed line) was detectable, and no signs of the IBS could be found on radiography. (d) In the control group 1 month after treatment, the radiographs showed signs of trabecular bone ingrowth (green asterisk) within the peripheral regions of the former defect area (red dashed line). However, the majority of the defect area was free of bone. (e) Three months after treatment, only a few trabecular bone structures (green asterisks) were found within the control defect area (red dashed line), whereas the center of the defect area still lacked bone. (f) Six months after treatment, a rudimentary trabecular network (green asterisks) was detectable within the former defect area (red dashed line) but was not comparable to the dense structure of the neighboring cancellous bone tissue of the femoral head or the trabecular structure found in the group treated with the IBS at this time point.

Histology of the bone defect treated with the injectable bone substitute (IBS) 1 month after implantation. (a) Overview of the implantation bed (red dashed line) of the IBS with an outer (OR) and inner implant region (IR). Within the OR, a high osteoconductive ingrowth of calcified bone tissue (red asterisks) was seen extending from the surrounding bone tissue (BT) towards the defect center. Within the IR, only low amounts of calcified bone tissue were visible (red arrows) (CT = connective tissue) (“total scan,” toluidine blue staining, magnification ×100). (b) Newly formed calcified bone tissue (NB) is present in the outer region of the implantation bed of the IBS. The β-TCP particles (TCP) of the bone substitute were found integrated within the trabecular bone tissue (orange arrows) and within the surrounding connective tissue (CT) (toluidine blue staining, magnification ×200, scale bar = 100 μm). (c) Growth of uncalcified bone tissue (blue asterisks) is observed in the inner implant regions, and is mainly related to the interstices of the β-TCP particles (TCP). Groups of ceramic particles (blue arrows) are present as a scaffold structure (toluidine blue staining, magnification ×200, scale bar = 100 μm). (d) Tissue reaction to the IBS in the inner implant region. β-TCP particles were found embedded in a vessel-rich connective tissue (CT) surrounded by mononuclear (green arrows) and multinuclear cells (yellow arrows) (vessels = black arrows) (toluidine blue staining, magnification ×400, scale bar = 10 μm).

Histology in the group treated with the injectable bone substitute (IBS) 3 months after implantation. (a) Overview of the implantation bed (red dashed line) of the IBS. The former outer implant region (OR) as well as the former inner region (IR) now contain high amounts of newly formed dense bone tissue (red asterisks) with a bulk-like trabecular organization. Nonintegrated biomaterial in this bone tissue was surrounded by connective tissue (blue asterisks) (CT = connective tissue, BT = neighbored bone tissue) (“total scan,” toluidine blue staining, magnification ×100). (b) This image shows the newly built bone tissue (NB) with its lamellar architecture, in which high amounts of the β-TCP particles (orange arrows) of the IBS are embedded (CT = connective tissue) (toluidine blue staining, magnification ×200, scale bar = 100 μm). (c) Shown are tissue reactions to the β-TCP particles (TCP) that were not involved in bone growth. These particles were embedded within a connective tissue (CT), and mononuclear (green arrows) and multinucleated cells (yellow arrows) were detectable at the surfaces of the particles (toluidine blue staining, magnification ×400, scale bar = 10 μm).

Histology of the bone healing mediated by the the injectable bone substitute (IBS) after 6 months. (a) Overview of a cross-section of the distal femur head. A trabecular network (red asterisks) was identifiable within the former defect area (red dashed line), and this network did not differ in its trabecular architecture and distribution compared with the surrounding healthy tissue (CT = connective tissue) (“total scan,” toluidine blue staining, magnification ×100). (b) Only very low amounts of the β-TCP particles (orange arrows) of the IBS were found embedded within the trabeculae of the bone tissue (BT). Furthermore, a growth of uncalcified bone tissue (blue asterisks) was observable along the scattered accumulations of β-TCP particles (white arrow) within the former outer region of the implantation bed of the IBS (CT = connective tissue) (toluidine blue staining, magnification ×200, scale bar = 100 μm). (c) Only low amounts of the β-TCP particles (TCP) within the former implant area were embedded within connective tissue (CT) surrounded by mononuclear (green arrows) and multinuclear cells (yellow arrows) (toluidine blue staining, magnification ×400, scale bar = 10 μm).

Postoperative course of the control defects in representative histological images. (a and b) The defect area (red dashed line in a) 1 month after the surgical treatment is shown. A low-grade growth of bone trabeculae (red asterisks) was visible starting from the bordering bone tissue (BT in a) of the defect areas, whereas only very few trabeculae (red arrows in a) were found within the defect center. In addition to the bone trabeculae (red asterisks), adipose cells/tissue (black arrows in b) and islands of connective tissue (green arrows in b) with mainly macrophages and granulocytes were detectable within the defect areas. (c and d) Histological images of the defect areas (red dashed line in c) 3 months after surgery are shown. Only a few newly built bone trabeculae (red asterisks in c) were found at the borders of the defect area (red asterisks in c) growing out from the neighboring bone tissue (BT in c) and within the defect centers (red arrows in c) at this time point. The soft tissue within the defect areas was mainly composed of adipose cells (blue asterisks in d). (e and f) These images are representative of the tissue structure 6 months after surgical intervention. The overview of the defect area (red dashed line in e) showed only a very low presence of trabecular bone tissue (red arrows). The former defect area contained mainly adipose tissue (blue asterisks in f) (toluidine blue stainings; a, c, and e: excerpt of “total scans,” magnification ×100; b, d, and f: magnification ×200, scale bars = 100 μm).

Results of the histomorphometrical measurements: (a) bone volume (in %) and (b) trabecular thickness (in μm) (*P > .05).