**3. Intercalary structural bone grafts**

Observations regarding 32 cases of structural bone grafts used for large osteoperiosteal gaps are summarized in **Table 4**. The largest group was 28 cases of giant cell tumors, 40% of these obtained uneventful success (**Figure 5**), 38% needed supplementary operation in the form of


**Table 4.** Large osteoperiosteal gaps (32) and structural grafts (1979–1999).

further autologous bone grafts for areas of non-union at host graft junctions or for pseudarthrosis in the intermediate part of the graft 20%, for control of infection, or for a combination of these factors (18%).

Six patients were considered a failure because the reconstruction failed. Two had recurrence of tumor, one had uncontrolled infection, and these ended up in amputations. In three patients despite two attempts at supplementary grafting, the areas of pseudarthrosis did not heal; these patients accepted an orthosis till further decision. One case of malignant fibrous histiocytoma failed because of recurrence of tumor within 4 months of limb salvage attempt.

Of the 11 patients of posterior or posterolateral spinal fusion, 10 were considered to have obtained satisfactory osseous fusion based upon clinical assessment and stress X-rays done 12–24 months after the operation (**Figure 6**). One young nurse who had posterior fusion along with Steffi's fixation at L3–L4 for spondylolisthesis was considered a failure because of the implant breakage observed 2 years after operations.

#### **3.1. Cytological and histological observation**

infection (three cases) and massive recurrence of tumor (three cases). All patients of solitary osseous lesions healed successfully. Polyostotic fibrous dysplasia (4), multiple fibrous defects (6), enchondromas (3), chondromyxoid fibroma (2), and enchondromatosis (2) by their biological nature may need reoperation for an increase in the size of the lesions which were insignificant at the time of the first surgery. One patient of fibrous dysplasia had to undergo a second operation for a new lesion, and one patient of enchondromatosis is awaiting surgery

**Figure 4.** A case of polyostotic fibrous dysplasia. The cyst in the trochanteric area remained healed after curettage and decal-bone grafting. After about 14 years of gap, another cyst in the supra-acetabular region needed treatment.

**Figure 3.** A recurred giant cell tumor of distal femur with pathological fracture. The healed status achieved by

Observations regarding 32 cases of structural bone grafts used for large osteoperiosteal gaps are summarized in **Table 4**. The largest group was 28 cases of giant cell tumors, 40% of these obtained uneventful success (**Figure 5**), 38% needed supplementary operation in the form of

for the additional area (**Figure 4**).

**3. Intercalary structural bone grafts**

intralesional curettage and decal-bone grafting following 12 years.

48 Bone Grafting - Recent Advances with Special References to Cranio-Maxillofacial Surgery

Fine-needle aspiration cytology (FNAC) was done from the perigraft region (in 20 patients) between the 10th and 40th day after grafting. The FNAC showed high cellularity composed of

**Figure 5.** Intercalary reconstruction after en bloc resection of giant cell tumor of distal femur. Note gradual incorporation and remodeling as observed in the 12-year follow-up.

agent. Its viability however depends upon the influence of processes of allograft preparation

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**Bone graft incorporation:** The biological process of incorporation of bone grafts is practically similar to that of a fracture healing. Under favorable environment, the following major steps occur in a cascadal fashion from the time of placement of the bone graft in the recipient bed to its incorporation and remodeling according to Wolff's law: (i) hematoma formation and its organization by invasion of neocapillaries surrounded by perivascular pluripotent mesenchymal cells; (ii) osteoclastic and phagocytic resorption of nonviable mineral (calcium hydroxyapatite), cellular debris and marrow fat, and tunneling of the graft-making channels for ingrowth and propagation of neocapillaries and osteoprogenitor cells; (iii) conversion (tissue engineering) of osteoprogenitor cells to osteoblastic cells under the influence of local osteoinductive agents (bone morphogenetic protein, other inductive agents, and growth factors present in the organic matrix of the bone graft) and the platelets. Laying down of the new bone (neo-osteogenesis) on the surface of matrix framework and along the vascular spaces/channels; and (iv) remodeling of the newly formed bone to conform to the trabecular pattern along the lines of functional loading and stress (according to Wolff's law). These events are a slow process; the grafted area needs protection with repetitive physiological axial or functional loading. The most challenging clinical condition of structural (intercalary) bone grafting for large osteoperiosteal gaps in the lower limb may take 2–4 years for adequate incorporation permitting unprotected loading [11, 12]. The mechanical strength of the reconstruct is weak for 1 and 1/2 to 2 years, after which the strength increases by more neo-osteogenesis. The least time is taken in a cavitary pathology which offers a very large osteogenic bed and copious surface for intimate contact with the graft [13–15]. As incorporation takes place from periphery to the center, the time taken for large cavities and large grafts is correspondingly higher. In un-demineralized cortical (e.g., fibula) graft, 20–30% (deepest sector) of the grafted bone may never get incorporated; it may stay

By HCL decalcification we aimed at the removal of nearly 50% of mineral, thus providing adequate structural integrity. The said treatment removed all cell debris and fat providing opened-up channels for ready penetration of neocapillaries and perivascular mesenchymal cells. Acid demineralization also removed the mineral from the surface of the bone, along the vascular channels, and on the lacunar spaces, thus exposing the matrix for intimate contact with the invading perivascular tissues, and facilitated the interaction between the graft matrix (most active osteoinductive principle) and the pluripotent mesenchymal cells from the host. The technique used by us does not destroy the biological osteoinductive property of the bone

Immunogenicity of allogenic bone is now better understood. Fresh unmatched and untreated allogenic bone inevitably evokes an immune response in the host. The immune response in general is delayed and mild and develops slowly; however, it results in "unexplained" graft resorption and delay or failure in its incorporation. In clinical practice deep-freezing, freezing, freeze-drying, and irradiation are currently employed to reduce immunogenicity. Pure BMP from an allogenic source or even a xenogenic source is considered to have negligible immunogenicity. We feel that a simple treatment of allogenic bone by HCL decalcification and ethanol preservation practically eliminates the antigenic material (cells and debris) to permit unhindered incorporation in clinical practice as observed in our cases. Overall analysis

and preservation [5–10].

incarcerated surrounded by newly formed bone.

matrix.

**Figure 6.** A child having tuberculosis of the spine involving multiple vertebrae: posterior spinal fusion was performed using allogenic decal-bone. Note the incorporation of the graft with remodeling during a 3-year follow-up.

polymorphs, lymphocytes, and macrophages between 10 and 20 days. The cellularity gradually reduced with relative increase in the number of lymphocytes. By the 40th postimplantation day, the macrophages were practically absent, and one could see appreciable osteogenic activity by the presence of osteoblasts and osteoclasts. No cellular immune reaction was discernable.

Periodic core biopsy in early stages and biopsy of the graft in patients who required a second operation showed histological and tetracycline fluorescence evidence of neo-osteogenesis between 6 and 12 weeks. The fluorescence in the implanted allogenic bone was quantitatively the same as the bone of a patient's iliac crest in specimens available 12 months after the grafting.

### **3.2. Discussion**

**Contents of bone grafts and their roles:** In general calcium hydroxyapatite, the predominant mineral in bones provides an inert framework providing mechanical stability and offering a lattice work for penetration of neocapillaries, reparative tissues, and osteoconduction (**Table 5**). Only the most superficial bone-forming cells in fresh autografts which survive getting nutrition by tissue perfusion provide direct osteogenic activity. In allografts no viable cells are expected; however, the debris of dead cells act as the most potent immunogenic agent. Organic matrix provides the most potent bone morphogenetic (bone induction principles)


**Table 5.** Contents of bone graft and their role.

agent. Its viability however depends upon the influence of processes of allograft preparation and preservation [5–10].

**Bone graft incorporation:** The biological process of incorporation of bone grafts is practically similar to that of a fracture healing. Under favorable environment, the following major steps occur in a cascadal fashion from the time of placement of the bone graft in the recipient bed to its incorporation and remodeling according to Wolff's law: (i) hematoma formation and its organization by invasion of neocapillaries surrounded by perivascular pluripotent mesenchymal cells; (ii) osteoclastic and phagocytic resorption of nonviable mineral (calcium hydroxyapatite), cellular debris and marrow fat, and tunneling of the graft-making channels for ingrowth and propagation of neocapillaries and osteoprogenitor cells; (iii) conversion (tissue engineering) of osteoprogenitor cells to osteoblastic cells under the influence of local osteoinductive agents (bone morphogenetic protein, other inductive agents, and growth factors present in the organic matrix of the bone graft) and the platelets. Laying down of the new bone (neo-osteogenesis) on the surface of matrix framework and along the vascular spaces/channels; and (iv) remodeling of the newly formed bone to conform to the trabecular pattern along the lines of functional loading and stress (according to Wolff's law). These events are a slow process; the grafted area needs protection with repetitive physiological axial or functional loading. The most challenging clinical condition of structural (intercalary) bone grafting for large osteoperiosteal gaps in the lower limb may take 2–4 years for adequate incorporation permitting unprotected loading [11, 12]. The mechanical strength of the reconstruct is weak for 1 and 1/2 to 2 years, after which the strength increases by more neo-osteogenesis. The least time is taken in a cavitary pathology which offers a very large osteogenic bed and copious surface for intimate contact with the graft [13–15]. As incorporation takes place from periphery to the center, the time taken for large cavities and large grafts is correspondingly higher. In un-demineralized cortical (e.g., fibula) graft, 20–30% (deepest sector) of the grafted bone may never get incorporated; it may stay incarcerated surrounded by newly formed bone.

polymorphs, lymphocytes, and macrophages between 10 and 20 days. The cellularity gradually reduced with relative increase in the number of lymphocytes. By the 40th postimplantation day, the macrophages were practically absent, and one could see appreciable osteogenic activity by the presence of osteoblasts and osteoclasts. No cellular immune reaction was discernable. Periodic core biopsy in early stages and biopsy of the graft in patients who required a second operation showed histological and tetracycline fluorescence evidence of neo-osteogenesis between 6 and 12 weeks. The fluorescence in the implanted allogenic bone was quantitatively the same as the bone of a patient's iliac crest in specimens available 12 months after the grafting.

**Figure 6.** A child having tuberculosis of the spine involving multiple vertebrae: posterior spinal fusion was performed

using allogenic decal-bone. Note the incorporation of the graft with remodeling during a 3-year follow-up.

50 Bone Grafting - Recent Advances with Special References to Cranio-Maxillofacial Surgery

**Contents of bone grafts and their roles:** In general calcium hydroxyapatite, the predominant mineral in bones provides an inert framework providing mechanical stability and offering a lattice work for penetration of neocapillaries, reparative tissues, and osteoconduction (**Table 5**). Only the most superficial bone-forming cells in fresh autografts which survive getting nutrition by tissue perfusion provide direct osteogenic activity. In allografts no viable cells are expected; however, the debris of dead cells act as the most potent immunogenic agent. Organic matrix provides the most potent bone morphogenetic (bone induction principles)

**3.2. Discussion**

Ca hydroxyapatite Mechanical stability Autogenous surviving cells Osteogenesis Allogenic non-surviving cells Immunogenesis

**Table 5.** Contents of bone graft and their role.

Matrix Bone morphogenetic agents(weak mechanical strength)

By HCL decalcification we aimed at the removal of nearly 50% of mineral, thus providing adequate structural integrity. The said treatment removed all cell debris and fat providing opened-up channels for ready penetration of neocapillaries and perivascular mesenchymal cells. Acid demineralization also removed the mineral from the surface of the bone, along the vascular channels, and on the lacunar spaces, thus exposing the matrix for intimate contact with the invading perivascular tissues, and facilitated the interaction between the graft matrix (most active osteoinductive principle) and the pluripotent mesenchymal cells from the host. The technique used by us does not destroy the biological osteoinductive property of the bone matrix.

Immunogenicity of allogenic bone is now better understood. Fresh unmatched and untreated allogenic bone inevitably evokes an immune response in the host. The immune response in general is delayed and mild and develops slowly; however, it results in "unexplained" graft resorption and delay or failure in its incorporation. In clinical practice deep-freezing, freezing, freeze-drying, and irradiation are currently employed to reduce immunogenicity. Pure BMP from an allogenic source or even a xenogenic source is considered to have negligible immunogenicity. We feel that a simple treatment of allogenic bone by HCL decalcification and ethanol preservation practically eliminates the antigenic material (cells and debris) to permit unhindered incorporation in clinical practice as observed in our cases. Overall analysis in our clinical material has been approximately 80–90% successful for benign cavitary lesions; for impaction grafts in revision joint surgeries, 50–70% success for structural reconstruction in circumferential osteoperiosteal gaps; and 70–90% clinical success in extensive spinal fusion. Supplementary procedures for obtaining success in difficult cases, especially for osteoperiosteal gaps, are an accepted norm in 20–30% of cases. The success rate in our clinical cases is compatible with the observations of the outcome where allogenic bone was used from more sophisticated bone banks. Allogenic bone graft is a rational option when the recipient's patient owns bones that are inherently defective (e.g., fibrous dysplasia, enchondromatosis).

[3] Tuli SM, Singh AD. The osteoinductive property of decalcified bone matrix: An experi-

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