**4. Biological characteristics**

Bone tissue basically consists of an organic matrix of collagen type I, containing low molecular weight proteoglycans and non-collagen proteins, a mineral part (mainly hydroxyapatite) and water, corresponding to 25%, 65% and 10% of the bone weight, respectively.

Despite the great power for repair, the bone does not always respond appropriately when affected by extensive osteolysis. Because of these situations, extremely frequent in orthopaedic surgery, especially in RTHAs, there is eagerness to search for high quality bone grafts and other biomaterials that can fill these gaps and restore availabilty.

Bone grafts can be of cortical, cancellous or cortico-cancellous types, depending on the site of origin and can be used in blocks, segments or morselised. These different types of bone grafts will provide distinct mechanical and biological responses. For instance, the cortical bone is less osteogenic than the spongy bone; showing however, a higher structural quality, for long periods and even in the absence of adequate integration. The greater osteogenic features of cancellous bone (Figures 1a, 1b and 1c) has been recognised for more than 40 years, which has stimulated its use in many situations, being crucial for use in RTHAs.

After the transplant of a spongy bone there is a sequence of histological events that starts with inflammatory response, macrophage invasion, neovascularization and differentiation of mesenchymal cells into osteoblasts that places an osteoid layer in a necrotic trabecular bone remainings. Viable nuclei are reabsorbed by osteoblasts and the matrix is eventually replaced by necrotic trabeculae of newly formed bone. All these events are probably mediated by inducing protein factors.

The Biology of Bone Grafts 239

Fig. 1b. Revision of femoral component left with bone graft and bovine freeze-dried,

morcelised impacted. 60 months of evolution.

Fig. 1a. Revision as of right femoral component with bone graft and frozen morcelised impacted. 70 months of evolution.

Fig. 1a. Revision as of right femoral component with bone graft and frozen morcelised

impacted. 70 months of evolution.

Fig. 1b. Revision of femoral component left with bone graft and bovine freeze-dried, morcelised impacted. 60 months of evolution.

The Biology of Bone Grafts 241

Fig. 2a. Biopsy bone grafting with bovinefreeze-dried three years after surgery. White arrow

In a classic study, Urist described ectopic bone formation after intramuscular implantation of demineralised bovine bone matrix in rabbits and rats. This discovery was crucial in the biomaterial and graft fields and supported the search for substances capable of inducing cell differentiation present in bone matrix. Subsequent investigations, led by Urist demonstrated that low molecular weight proteins could be extracted from demineralised bone matrix, having great osteogenic activity and being called bone morphogenetic proteins (BMPs), and belonging to a superfamily of proteins called transforming growth factors beta (TGF-ß) responsible for inducing growth. The superfamily of TGF-ß proteins regulates many biological processes including cell growth, differentiation and embryo formation. BMPs have been shown to be important regulators in the development and regeneration of skeletal tissue having the BMP2 and BMP7 the highest osteoinductive potential. Another important factor is the reaction triggered by antigenic grafts. The antigens present into the graft cells probably play a very important role on the outcomes of bone transplants. It seems clear that these reactions are not mediated by T or B lymphocytes, but by other cells in the bone marrow. In vitro evidences suggest that the granulocyte lineages in the bone marrow are responsible for triggering immune responses and removal of bone marrow cells therefore and can be directly related

Fresh, frozen and lyophilized bone grafts were experimentally compared in rabbits from the immunological point of view. The first two grafts caused serologically detectable immune response, while the third one, highly purified (free of fat and marrow cells) was not able to

= graft; black arrow = new bone

to a decrease in graft immune response.

immunologically sensitize animals.

Fig. 1c. Revision of the acetabular component with lyophilized bovine bone graft and graft block frozen morcelised impacted. 82 months of evolution.

Histologic assessment was performed and described by Buma et al. in eight patients who underwent revision of the acetabular component after previous operation with impacted bone grafting technique. All but one, that has not even shown revascularization, samples revealed different stages of integration depending on the time elapsed following implantation. At 4 months, there was an established revascularization, osteoblasts replacing parts of the implant and presence of a small graft and new bone formation. Samples with a longer evolution showed the graft replaced with new bone. Another sample with 28 months in contact with the cement layer revealed viable bone presenting, however, a predominantly fibrous tissue interface5. Similar results on the femoral component were demonstrated in another study published by Ullmark and Obrant in 2002 and by Galia in acetabular biopsies in patients with traumatic dislocation of RTHA (Figures 2a and 2b) 3 years after the first revision procedure.

Fig. 1c. Revision of the acetabular component with lyophilized bovine bone graft and graft

Histologic assessment was performed and described by Buma et al. in eight patients who underwent revision of the acetabular component after previous operation with impacted bone grafting technique. All but one, that has not even shown revascularization, samples revealed different stages of integration depending on the time elapsed following implantation. At 4 months, there was an established revascularization, osteoblasts replacing parts of the implant and presence of a small graft and new bone formation. Samples with a longer evolution showed the graft replaced with new bone. Another sample with 28 months in contact with the cement layer revealed viable bone presenting, however, a predominantly fibrous tissue interface5. Similar results on the femoral component were demonstrated in another study published by Ullmark and Obrant in 2002 and by Galia in acetabular biopsies in patients with traumatic dislocation of RTHA

block frozen morcelised impacted. 82 months of evolution.

(Figures 2a and 2b) 3 years after the first revision procedure.

Fig. 2a. Biopsy bone grafting with bovinefreeze-dried three years after surgery. White arrow = graft; black arrow = new bone

In a classic study, Urist described ectopic bone formation after intramuscular implantation of demineralised bovine bone matrix in rabbits and rats. This discovery was crucial in the biomaterial and graft fields and supported the search for substances capable of inducing cell differentiation present in bone matrix. Subsequent investigations, led by Urist demonstrated that low molecular weight proteins could be extracted from demineralised bone matrix, having great osteogenic activity and being called bone morphogenetic proteins (BMPs), and belonging to a superfamily of proteins called transforming growth factors beta (TGF-ß) responsible for inducing growth. The superfamily of TGF-ß proteins regulates many biological processes including cell growth, differentiation and embryo formation. BMPs have been shown to be important regulators in the development and regeneration of skeletal tissue having the BMP2 and BMP7 the highest osteoinductive potential. Another important factor is the reaction triggered by antigenic grafts. The antigens present into the graft cells probably play a very important role on the outcomes of bone transplants. It seems clear that these reactions are not mediated by T or B lymphocytes, but by other cells in the bone marrow. In vitro evidences suggest that the granulocyte lineages in the bone marrow are responsible for triggering immune responses and removal of bone marrow cells therefore and can be directly related to a decrease in graft immune response.

Fresh, frozen and lyophilized bone grafts were experimentally compared in rabbits from the immunological point of view. The first two grafts caused serologically detectable immune response, while the third one, highly purified (free of fat and marrow cells) was not able to immunologically sensitize animals.

The Biology of Bone Grafts 243

1991, studied the histological and biomechanical properties of different ways of bone graft processing in rats and noted that an increased stiffness of the bone after freezing at -80°C and lyophilising it. The freeze-dried demineralised bone has initially decreased its mechanical strength. However, after 16 weeks, a progressive increase in resistance of the grafts was observed and was likely related to its biological interaction, which might be an indicator of the osteoinductive properties of the graft. They also concluded that on the

The physical properties of human and bovine trabecular bone are documented and their results are available, however, the range of dispersion is very wide. The Young's module, for example, in one study ranged from 70 to 673 MPa and compressive strength varied between 2.44 and 6.24 MPa. This dispersion occurred for both human and bovine bone and may be related to several factors such as donor age, bone density and methodology used in

Cornu et al. in 2001, has demonstrated in vitro that the lyophilised morcelised and impacted bone is mechanically superior to the morcelised and impacted deep-frozen bone since, at least, has the same resistance after impaction, that is achieved however, more quickly and with fewer impacts, and the authors assumed as to the fact that lyophilised material was devoid of fat and bone marrow. Moreover, Macedo et al. in 1999, using an automated compression machine compared in vitro, the compressive strength of frozen and freezedried bovine bone rehydrated for an hour and found out that deep-frozen bovine bone grafts after defrosting, has similar compressive loads and deformation rate of the rehydrated

Another extremely important issue is the study of methods of sterilization of frozen and freeze-dried grafts, since there still remains controversy and need for further studies. The currently available techniques have advantages and disadvantages regarding efficacy and maintenance of mechanical and biological properties. Significant deleterious effects on the use of cobalt 60 in the sterilisation of freeze-dried grafts have been reported, demonstrating that, even at low dosage, radiation is capable of destroying the morphogenetic properties,

The effects of radiation on the biomechanics of the grafts are dose-dependent. Fidele et al. in 1995, studied the damage caused by the application of different doses of gamma radiation exposure on seven biomechanical parameters of frozen allogenic patellar bone-grafting. The sterilization dose accepted for inactivation of HIV, for example, is about 25 kGy. However, four out of the seven parameters measured were reduced after 20 kGy and after 30 kGy all parameters have shown significant reductions. Also, Zhang, Cornu and Delloye in 1997, in an experimental study in rats, compared the ability of graft osteoinduction after gamma radiation sterilisation (25 kGy), ethylene oxide (EO) at 55°C and 40°C or preservation in ethanol showing that the OE at 40°C and ethanol have not negatively affected the osteoinductive capacity, gamma radiation has decreased 40% and OE to 55°C had an almost completely loss of this potential. Some authors indicated that other factors in sterilisation need to be observed are the toxic residues from the OE that may remain in the graft and are released when in contact with liquids. Reference is also considered to the toxicity of gamma radiation when in contact with fat present in the graft, in addition to the mechanical change

features of integration, as expected, the autologous bone showed the best results.

**6. Methods sterilization, infectious diseases and biosafety** 

the study.

lyophilised bovine bone.

mainly in non-demineralised bone.

that the radiation may cause.

Fig. 2b. Biopsy of frozen human bone graft. 36 months of evolution. White arrow = graft; black arrow = new bone.

Although the freeze-dried grafts, whether human or bovine, are available in many medical centres worldwide, most work on RTHAs refers to the use of frozen bone grafts in blocks or cortical, but especially, most recently the spongy morcelised and impacted.

Tagil, in his PhD thesis published in 2000, attempted to explain the reasons for succeeding using the technique of frozen morcelised and impacted bone, once theoretically, the large volume of necrotic bone exposed to great mechanical stress tend to collapse, as in the vascular necrosis of the femoral head or knee. After detailed study, Tagil found out the following possibilities: (a) morcelised bone, as in a comminuted fracture, would produce extensive surface contact allowing access and release of biologically active substances; (b) impaction may improve osteoconductive properties of the graft leading to the release of BMPs and this way favouring osteointegration and; (c) the high elasticity may allow small deformations that would stimulate new bone formation. The importance of this study lays in the fact that it is essential, from a scientific standpoint, to know the pros and cons of a due technique and why results may be good or bad, to better use and indicate such a procedure more securely and confidentially.
