**4. Clinical application of grafts over the years**

The use of bone tissue for replacement of bone losses is not a recent procedure. Since last century there have been accounts of the use of these tissues in humans and in experiments with animals as a means of assessing their efficacy.

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 41

understood that part of this capacity is the responsibility of the superfamilies of proteins (TGF), including the morphogenetic protein (BMP) present in the bone matrix. (Malafaya et

The decade of 1980 was marked by the advent of the acquired immunodeficiency syndrome (AIDS) that gives rise to discussions on the safety of the clinical use of homologous tissues. The biological risk of disease transmission between tissue donors and recipients is the topic of greatest relevance and importance in the period. The tissue banks existing at that time were encouraged to review donor selection protocols with the objective of avoiding the transmission of these diseases. This encouragement was provided mainly by the international public health regulatory agencies, such as the FDA (Food and Drug Administration) and other institutions related to the Haemovigilance and tissue transplantation systems. The result was a standardization of the internal processes of banks with respective preparation of standards by the main global tissue bank associations (American Associating of Tissue Banks - AATB and European Association of Tissue Banks-EATB), which contributes to the gain of quality of tissue made available by these services

With the availability of more reliable grafts by the tissue banks, their use, biological behavior and indication by surgeons become a viable treatment option. (Santos, 2011).

Similar to the repair process in fractures and in the development of the musculoskeletal system, the osseointegration of grafts takes place after a selection of primordial cells that are differentiated into osteoblasts under the influence of osteogenic factors. (Thies et al., 1992). The main objective expected in the use of grafts is the ability to selectively induce the primordial events of the integration process, such as osteoinduction, osteoconduction and

According to Tomford and Mankin (1999), for cortical bone grafts to be incorporated into the receptor bed, there must be revascularization of this bed. When this process does not occur, the repair area loses balance in resorption and the graft might suffer fatigue fractures.

Boldt et al. (2001) evaluate the use of frozen bone graft in 173 acetabular reconstructions and 79 femoral reconstructions in humans. Femoral heads obtained from a local bone bank are particulated and impacted in the faults. After a mean follow-up period of four years, they report acetabular clinical stability in 97.2 % of the cases, graft incorporation in 74% in the acetabula and 61% in the femurs, according to radiological analysis. They conclude that the results obtained with the use of impacted grafts are promising, except for the reconstruction

Janssen et al. (2001) studied the use of homologous cortical rings obtained from femoral diaphysis for reconstruction of intervertebral discs of the lumbar spine in 137 patients. The results show that arthrodesis was achieved in 94% of the cases and they do not report signs

Weyts et al. (2003) analyze samples of femoral heads collected after the primary arthroplasty of two human donors. The tissues are cryopreserved at - 80 °C for a minimum period of six months, according to the protocols of the American and European Tissue Bank Associations. After this period, these tissues are biopsied and submitted to cell culture (for survival observation) and PCR (polymerase chain reaction) for genetic screening. The results show

of type III acetabular defects, where a reinforcement cage is recommended.

the presence of live cells belonging to the donors in the analyzed samples.

(Nather, 1991; Galea, Kearney, 2005; Santos, 2007).

osteogenesis (Lindhe et al., 1997).

of resorption.

Spongy grafts are consolidated more quickly.

al, 2002).

In these studies, many treatments with the use of bone grafts of autologous and homologous nature have been proposed over the years. The first homologous bone transplant is described by William MacEwen in 1878 (Giovani, 2005). At this time the treatment of osteomyelitis was performed by means of surgical resections of the infected segments. Homologous tibial segments (obtained from patients submitted to osteotomies) were used in the reconstruction of a bone defect caused by the resection of part of the humerus of a young man suffering from osteomyelitis (Tomford, 2000). Encouraging results of the consolidation of the bone graft with the receptor bed (MacEwen, 1909) motivated researchers back then. In this period, there was no consensus about which bones can specifically be used for transplantation. Tissues were obtained randomly from donors that were victims of fractures, resections and amputations. For the storage and processing of these tissues, professionals used protocols and equipment that today are not the best suited to this purpose (Tomford, Mankin, 1999; Tomford, 2000). Nowadays a large portion of these studies has only historical value with respect to the pioneer spirit of these researchers.

The clinical use of allografts during this period was in low demand, and occurred on an experimental basis at some centers from all around the world. With the availability of antibiotic therapy, changes occurred in the indication of these tissues, and patients with osteomyelitis were then submitted to pharmacological treatments, to the detriment of surgical procedures (Tomford, 2000). Thus surgical resections of infected segments cease to be a priority, and allografts are then used in the reconstructions of bone defects caused by tumor resections. This change causes the studies from the time to evolve as well, and to give more detailed accounts of the cellular processes involved in the osseointegration of grafts. The consensuses of these first studies serve as guidelines for the first grafts performed at that time.

The use of cryopreserved allografts presents some advantages over autologous tissue, such as the availability of the necessary quantity of tissue and a decrease in postoperative morbidity. As regards morphology, there are some differences in the vascularization process of the cortical and spongy bone grafts. In the cortical bone, the repair is started by the action of osteoclasts and in the spongy bone, by the osteoblasts. Another difference lies in the revascularization time, which is slower for cortical bone and faster for spongy bone.

At that time, Urist (1965) was already describing that the osteoprogenitor cells responsible for the bone repair process are derived from monocytes, present in an elevated number in the repair zone coming from the bone marrow, and that the osseointegration of grafts is achieved, since primitive cells not yet differentiated can differentiate into viable osteoblasts from osteoinductive substances. Perivascular mesenchymal cells disaggregate and migrate to the grafting area, where they reaggregate, proliferate and differentiate to form new bone. (Urist et al, 1983)

Some substances secreted by certain cell types interfere in or even modulate the cellular processes of osseointegration, today known as Cytokines or growth factors.

Urist made important discoveries in this area back in 1965, even proposing a new bone processing procedure aimed at the removal of a calcified layer (demineralization) from the matrix, making this graft more osteoinductive. At the time this kind of tissue was called Demineralized Bone Matrix (DBM), and the name is still in use today. Later, it was

In these studies, many treatments with the use of bone grafts of autologous and homologous nature have been proposed over the years. The first homologous bone transplant is described by William MacEwen in 1878 (Giovani, 2005). At this time the treatment of osteomyelitis was performed by means of surgical resections of the infected segments. Homologous tibial segments (obtained from patients submitted to osteotomies) were used in the reconstruction of a bone defect caused by the resection of part of the humerus of a young man suffering from osteomyelitis (Tomford, 2000). Encouraging results of the consolidation of the bone graft with the receptor bed (MacEwen, 1909) motivated researchers back then. In this period, there was no consensus about which bones can specifically be used for transplantation. Tissues were obtained randomly from donors that were victims of fractures, resections and amputations. For the storage and processing of these tissues, professionals used protocols and equipment that today are not the best suited to this purpose (Tomford, Mankin, 1999; Tomford, 2000). Nowadays a large portion of these studies has only historical value with respect to the pioneer spirit of these researchers.

The clinical use of allografts during this period was in low demand, and occurred on an experimental basis at some centers from all around the world. With the availability of antibiotic therapy, changes occurred in the indication of these tissues, and patients with osteomyelitis were then submitted to pharmacological treatments, to the detriment of surgical procedures (Tomford, 2000). Thus surgical resections of infected segments cease to be a priority, and allografts are then used in the reconstructions of bone defects caused by tumor resections. This change causes the studies from the time to evolve as well, and to give more detailed accounts of the cellular processes involved in the osseointegration of grafts. The consensuses of these first studies serve as guidelines for the first grafts performed at

The use of cryopreserved allografts presents some advantages over autologous tissue, such as the availability of the necessary quantity of tissue and a decrease in postoperative morbidity. As regards morphology, there are some differences in the vascularization process of the cortical and spongy bone grafts. In the cortical bone, the repair is started by the action of osteoclasts and in the spongy bone, by the osteoblasts. Another difference lies in the

At that time, Urist (1965) was already describing that the osteoprogenitor cells responsible for the bone repair process are derived from monocytes, present in an elevated number in the repair zone coming from the bone marrow, and that the osseointegration of grafts is achieved, since primitive cells not yet differentiated can differentiate into viable osteoblasts from osteoinductive substances. Perivascular mesenchymal cells disaggregate and migrate to the grafting area, where they reaggregate, proliferate and differentiate to form new bone.

Some substances secreted by certain cell types interfere in or even modulate the cellular

Urist made important discoveries in this area back in 1965, even proposing a new bone processing procedure aimed at the removal of a calcified layer (demineralization) from the matrix, making this graft more osteoinductive. At the time this kind of tissue was called Demineralized Bone Matrix (DBM), and the name is still in use today. Later, it was

processes of osseointegration, today known as Cytokines or growth factors.

revascularization time, which is slower for cortical bone and faster for spongy bone.

that time.

(Urist et al, 1983)

understood that part of this capacity is the responsibility of the superfamilies of proteins (TGF), including the morphogenetic protein (BMP) present in the bone matrix. (Malafaya et al, 2002).

The decade of 1980 was marked by the advent of the acquired immunodeficiency syndrome (AIDS) that gives rise to discussions on the safety of the clinical use of homologous tissues. The biological risk of disease transmission between tissue donors and recipients is the topic of greatest relevance and importance in the period. The tissue banks existing at that time were encouraged to review donor selection protocols with the objective of avoiding the transmission of these diseases. This encouragement was provided mainly by the international public health regulatory agencies, such as the FDA (Food and Drug Administration) and other institutions related to the Haemovigilance and tissue transplantation systems. The result was a standardization of the internal processes of banks with respective preparation of standards by the main global tissue bank associations (American Associating of Tissue Banks - AATB and European Association of Tissue Banks-EATB), which contributes to the gain of quality of tissue made available by these services (Nather, 1991; Galea, Kearney, 2005; Santos, 2007).

With the availability of more reliable grafts by the tissue banks, their use, biological behavior and indication by surgeons become a viable treatment option. (Santos, 2011).

Similar to the repair process in fractures and in the development of the musculoskeletal system, the osseointegration of grafts takes place after a selection of primordial cells that are differentiated into osteoblasts under the influence of osteogenic factors. (Thies et al., 1992). The main objective expected in the use of grafts is the ability to selectively induce the primordial events of the integration process, such as osteoinduction, osteoconduction and osteogenesis (Lindhe et al., 1997).

According to Tomford and Mankin (1999), for cortical bone grafts to be incorporated into the receptor bed, there must be revascularization of this bed. When this process does not occur, the repair area loses balance in resorption and the graft might suffer fatigue fractures. Spongy grafts are consolidated more quickly.

Boldt et al. (2001) evaluate the use of frozen bone graft in 173 acetabular reconstructions and 79 femoral reconstructions in humans. Femoral heads obtained from a local bone bank are particulated and impacted in the faults. After a mean follow-up period of four years, they report acetabular clinical stability in 97.2 % of the cases, graft incorporation in 74% in the acetabula and 61% in the femurs, according to radiological analysis. They conclude that the results obtained with the use of impacted grafts are promising, except for the reconstruction of type III acetabular defects, where a reinforcement cage is recommended.

Janssen et al. (2001) studied the use of homologous cortical rings obtained from femoral diaphysis for reconstruction of intervertebral discs of the lumbar spine in 137 patients. The results show that arthrodesis was achieved in 94% of the cases and they do not report signs of resorption.

Weyts et al. (2003) analyze samples of femoral heads collected after the primary arthroplasty of two human donors. The tissues are cryopreserved at - 80 °C for a minimum period of six months, according to the protocols of the American and European Tissue Bank Associations. After this period, these tissues are biopsied and submitted to cell culture (for survival observation) and PCR (polymerase chain reaction) for genetic screening. The results show the presence of live cells belonging to the donors in the analyzed samples.

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 43

limitation of the quantity and variety of graft obtained (Smith et al. 1984; Cunningham,

This tendency, combined with the growing number of patients with bone loss that seek specialized orthopedic and dental services, leverages the creation of some Tissue Banks in

In Brazil, the standardization of Musculoskeletal Tissue Banks is linked to specific laws of our country. We have a General Coordination Office for the National Transplant System – SNT of the Ministry of Health that discusses and prepares, together with technical chambers, legislations involving the use of tissue by the medical and dental community. The guidelines are based on protocols already developed by some centers and on the standards of international associations (European Association of Tissue Banks - EATB and American Association of Tissue Banks-AATB). The creation of the first reference centers in the largescale capture, processing and distribution of musculoskeletal tissue and some of these experiences are described. (Amatuzzi et al., 2000, Amatuzzi et al., 2004). Tissue Banks in Brazil are controlled by the General Management of Blood, other Tissues, Cells and Organs - GGSTO of the National Health Surveillance Agency - ANVISA, an institution similar to the U.S. FDA. This agency focuses its activities on health surveillance and on quality control, traceability, appraisal of risks and of adverse effects involving tissue transplants in the country and its guidelines are published in the form of legislation5 that also defines Musculoskeletal Tissue Bank as "the service that, with physical facilities, equipment, human resources and adequate techniques, has as its duties the performance of clinical, laboratory and serological triage of tissue donors, the removal, identification, transportation, processing, storage and delivery of bones, soft tissues (cartilage, fasciae, serous membranes, muscle tissue, ligaments and tendons) and their derivatives, of human origin for therapeutic

The description of activities of a musculoskeletal tissue bank is summarized in the algorithm

Every activity related to the bank should also be based on the ethical principles inherent to

 **Autonomy and self-determination:** The recipient of tissue from the musculoskeletal system should be provided with information in accessible language about the entire tissue obtainment process, the risks and the chances of success or failure of the treatment. The following stage is the patient's decision, after their evaluation of the

Professionals with specific training provide all the required information, using language exempt from complex or technical terms, enabling the patient to achieve easy understand in order to make the final decision. For this document to be authentic, the consent must be free, that is, not caused by coercion. The professionals of a musculoskeletal tissue bank should be objective and impartial while providing

guidance to recipients. Every process is recorded in the Recipient's Form.

Reddi, 1992; Drumond, 2000).

the country in an experimental manner.

purposes, research and teaching".

below ( **Illustration 01**).

**6. Activities of a musculoskeletal tissue bank** 

the activities of any organ transplantation. They are:

5Administrative Ruling no. 211, of March 24, 2003

information received, set out in an informed consent form.

Lavernia et al. (2004) researched the adoption and use of allografts by orthopedic surgeons in 340 U.S. hospitals. The frozen graft from tissue banks certified by the American Association of Tissue Banks - AATB is used the most often by orthopedists for the treatment of knee and hip bone loss.

Schreurs et al. (2005) conduct a study to evaluate the use of homologous bone graft from a tissue bank in the reconstruction of 33 femoral defects. Bone graft impaction precedes the fixation of the femoral nail. After a minimum follow-up of eight years there is functional improvement of the joint according to the Harris hip score (from 49 to 85 points from pre- to postoperative period) and good survival according to the Kaplan-Meier method. Although four patients have had femoral fractures, the authors conclude that the graft impaction technique and use of cemented femoral nail results in excellent survival for eight to thirteen years.

Cabrita (2007) studies the treatment of infected hip arthroplasties with and without the use of the antibiotic-impregnated cement spacer. For the reconstruction of bone stock, Cabrita uses the massive particulated homologous graft in 60.9% of the patients treated and does not report complications related to their use.

In a review of concepts, Giannoudis et al. (2005) emphasize the advantages of the use of bone grafts in the area of orthopedics and traumatology. They describe the cellular events present in literature that involves the osseointegration process of autologous, homologous grafts and of biocompatible synthetic substitutes. They stress the osteoinductive characteristic of fresh grafts and the osteoconductive characteristic of frozen and lyophilized grafts. Heyligers and Kleim (2005) verify the presence of live cells with growth potential in samples of femoral heads cryopreserved at - 80 ºC, over a minimum period of six months. The authors stress the importance of discussing the osteoconductive potential of grafts and highlight the need to investigate the role of these surviving cells from the frozen tissue in the bone formation process after its implantation. Besides the bone cells, other cells and inflammatory factors play a vital role in the bone repair process. The macrophages and substances such as interleukin one, six, eleven (IL-1, IL-6, IL-11), RANKL and osteoprotegerin (OPG) are found during the first three days after the lesion (Gerstenfeld, Einhorn, 2006). As far as macrophages are concerned, Knighton et al. (1982) explain that these cells are present in the repair zone and are capable of producing growth factors, which, in turn, stimulate the neovascularization, proliferation and migration of other cell types, such as the fibroblasts.

#### **5. Evolution of musculoskeletal tissue banks in Brazil**

Considering the satisfactory results in the use of allografts obtained from multiple donors of organs and tissues with brain death besides studies showing revascularization, osteointegration and bone formation at sites of graft ( Barros Filho, et al., 1989; Croci et al., 2003, Zhang et al., 2004; Dallari et al., 2006, Bitar et al, 2010), an increasing number of orthopedic surgeons and dentists currently opt to use of homologous grafts in our country. This fact is corroborated by the disadvantages already known in the use of autologous tissues, such as the increase in postoperative morbidity, greater risk of infection inherent to the second surgical procedure required for their obtainment, risk of nerve lesion and

Lavernia et al. (2004) researched the adoption and use of allografts by orthopedic surgeons in 340 U.S. hospitals. The frozen graft from tissue banks certified by the American Association of Tissue Banks - AATB is used the most often by orthopedists for the treatment

Schreurs et al. (2005) conduct a study to evaluate the use of homologous bone graft from a tissue bank in the reconstruction of 33 femoral defects. Bone graft impaction precedes the fixation of the femoral nail. After a minimum follow-up of eight years there is functional improvement of the joint according to the Harris hip score (from 49 to 85 points from pre- to postoperative period) and good survival according to the Kaplan-Meier method. Although four patients have had femoral fractures, the authors conclude that the graft impaction technique and use of cemented femoral nail results in excellent survival for eight to thirteen

Cabrita (2007) studies the treatment of infected hip arthroplasties with and without the use of the antibiotic-impregnated cement spacer. For the reconstruction of bone stock, Cabrita uses the massive particulated homologous graft in 60.9% of the patients treated and does

In a review of concepts, Giannoudis et al. (2005) emphasize the advantages of the use of bone grafts in the area of orthopedics and traumatology. They describe the cellular events present in literature that involves the osseointegration process of autologous, homologous grafts and of biocompatible synthetic substitutes. They stress the osteoinductive characteristic of fresh grafts and the osteoconductive characteristic of frozen and lyophilized grafts. Heyligers and Kleim (2005) verify the presence of live cells with growth potential in samples of femoral heads cryopreserved at - 80 ºC, over a minimum period of six months. The authors stress the importance of discussing the osteoconductive potential of grafts and highlight the need to investigate the role of these surviving cells from the frozen tissue in the bone formation process after its implantation. Besides the bone cells, other cells and inflammatory factors play a vital role in the bone repair process. The macrophages and substances such as interleukin one, six, eleven (IL-1, IL-6, IL-11), RANKL and osteoprotegerin (OPG) are found during the first three days after the lesion (Gerstenfeld, Einhorn, 2006). As far as macrophages are concerned, Knighton et al. (1982) explain that these cells are present in the repair zone and are capable of producing growth factors, which, in turn, stimulate the neovascularization, proliferation and migration of other cell

Considering the satisfactory results in the use of allografts obtained from multiple donors of organs and tissues with brain death besides studies showing revascularization, osteointegration and bone formation at sites of graft ( Barros Filho, et al., 1989; Croci et al., 2003, Zhang et al., 2004; Dallari et al., 2006, Bitar et al, 2010), an increasing number of orthopedic surgeons and dentists currently opt to use of homologous grafts in our country. This fact is corroborated by the disadvantages already known in the use of autologous tissues, such as the increase in postoperative morbidity, greater risk of infection inherent to the second surgical procedure required for their obtainment, risk of nerve lesion and

of knee and hip bone loss.

not report complications related to their use.

types, such as the fibroblasts.

**5. Evolution of musculoskeletal tissue banks in Brazil** 

years.

limitation of the quantity and variety of graft obtained (Smith et al. 1984; Cunningham, Reddi, 1992; Drumond, 2000).

This tendency, combined with the growing number of patients with bone loss that seek specialized orthopedic and dental services, leverages the creation of some Tissue Banks in the country in an experimental manner.

In Brazil, the standardization of Musculoskeletal Tissue Banks is linked to specific laws of our country. We have a General Coordination Office for the National Transplant System – SNT of the Ministry of Health that discusses and prepares, together with technical chambers, legislations involving the use of tissue by the medical and dental community. The guidelines are based on protocols already developed by some centers and on the standards of international associations (European Association of Tissue Banks - EATB and American Association of Tissue Banks-AATB). The creation of the first reference centers in the largescale capture, processing and distribution of musculoskeletal tissue and some of these experiences are described. (Amatuzzi et al., 2000, Amatuzzi et al., 2004). Tissue Banks in Brazil are controlled by the General Management of Blood, other Tissues, Cells and Organs - GGSTO of the National Health Surveillance Agency - ANVISA, an institution similar to the U.S. FDA. This agency focuses its activities on health surveillance and on quality control, traceability, appraisal of risks and of adverse effects involving tissue transplants in the country and its guidelines are published in the form of legislation5 that also defines Musculoskeletal Tissue Bank as "the service that, with physical facilities, equipment, human resources and adequate techniques, has as its duties the performance of clinical, laboratory and serological triage of tissue donors, the removal, identification, transportation, processing, storage and delivery of bones, soft tissues (cartilage, fasciae, serous membranes, muscle tissue, ligaments and tendons) and their derivatives, of human origin for therapeutic purposes, research and teaching".
