**14.1 Histomorphometry**

Histomorphometry aims to analyze bone morphology and its components (measurements of volume, area, perimeter etc.). This technique is developed primarily for rock analysis, and is currently employed to analyze cellular behaviors of tissues starting from their structural conformity, expressed in thin slices on a slide. The histological reading and the definition of elements that compose the bone microtexture is the main goal of this procedure. The method can be manual, semiautomatic and automatic. A microscope coupled to a micrometer ruler is used in the first. In the semiautomatic version the microscope is coupled to a computer, which in turn uses software that allows users to record and to quantitatively analyze the images of a slide, seen through the microscope lenses, which are projected onto a digitizing board and drawn manually. The definition of each histological structure is performed by a professional. Finally, the automatic technique allows users to capture the images from the microscope using video cameras and the definition of each structure is determined by the actual computer, which automatically analyzes the coloration of each structure. Although the latter is the fastest technique, it is also the least sensitive. (Jorgetti, 2003; Aaron, Shore, 2003)

This technique permits the analysis of primary or static parameters (extension, number and distance) and the derived parameters, which are divided into structural (analyze bone structure) and kinetic (analyze the dynamics of the bone tissue).

The histomorphometric parameters are measured at 125x zoom. The choice of the analysis area should consider the contact surface of the receptor bone with the allograft.

A microscope equipped with objective, micrometer ruler, cursor, digitizing board and image analysis software is used to quantify the structural static, bone tissue formation and resorption histomorphometric parameters. (**Illustration 22**)

The static histomorphometric parameters are classified as:

#### STRUCTURAL PARAMETERS

Total area (**T.Ar** mm2): total area measured;


Illustration 14. Equipment used for histomorphometric analysis (microscope coupled to digitizing board and Osteomeasure® software)

#### FORMATION PARAMETER

58 Current Frontiers in Cryopreservation

bone grafts processed in a tissue bank with different methods (lyophilized, demineralized and radiated (ALD); mineralized frozen (ACM) besides autogenous grafts (AT) and blood clot (CG). In the Guided Bone Regeneration (GBR) technique, samples of the groups of grafts were placed in 32 cylinders fastened to the calvaria of 08 animals. After 13 weeks the cylinder fill rates (bone volume of the ALD group) were similar to the ACM and superior to the autogenous graft). Bone neoformation also occurs during the use of homologous grafts in maxillary sinus lifting surgery, besides affording lower morbidity levels (Viscioni et al, 2010). Hence it should be considered a valid alternative for the replacement of autologous

Some techniques can be used today in the analysis of interface sites between receptor and donor bone, which generate information capable of providing subsidies for a greater

Histomorphometry aims to analyze bone morphology and its components (measurements of volume, area, perimeter etc.). This technique is developed primarily for rock analysis, and is currently employed to analyze cellular behaviors of tissues starting from their structural conformity, expressed in thin slices on a slide. The histological reading and the definition of elements that compose the bone microtexture is the main goal of this procedure. The method can be manual, semiautomatic and automatic. A microscope coupled to a micrometer ruler is used in the first. In the semiautomatic version the microscope is coupled to a computer, which in turn uses software that allows users to record and to quantitatively analyze the images of a slide, seen through the microscope lenses, which are projected onto a digitizing board and drawn manually. The definition of each histological structure is performed by a professional. Finally, the automatic technique allows users to capture the images from the microscope using video cameras and the definition of each structure is determined by the actual computer, which automatically analyzes the coloration of each structure. Although the latter is the fastest technique, it is also the least sensitive. (Jorgetti,

This technique permits the analysis of primary or static parameters (extension, number and distance) and the derived parameters, which are divided into structural (analyze bone

The histomorphometric parameters are measured at 125x zoom. The choice of the analysis

A microscope equipped with objective, micrometer ruler, cursor, digitizing board and image analysis software is used to quantify the structural static, bone tissue formation and

area should consider the contact surface of the receptor bone with the allograft.

structure) and kinetic (analyze the dynamics of the bone tissue).

resorption histomorphometric parameters. (**Illustration 22**) The static histomorphometric parameters are classified as:

Total area (**T.Ar** mm2): total area measured;

**14. Prospects in the application of osseointegration investigation** 

grafts in patients submitted to implant therapy.

understanding of the osseointegration process.

**methodologies in grafting areas** 

**14.1 Histomorphometry** 

2003; Aaron, Shore, 2003)

STRUCTURAL PARAMETERS


Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 61

from the cellular surface and from the pericellular environment and some intracellular

This technique makes it possible to evaluate bone matrix proteins (type I collagen, osteocalcin, osteopontin and osteonectin) and the MMPs during the osseointegration

Peripheral quantitative computed tomography - pQCT is a method used to measure the appendicular skeleton that, besides furnishing a broad range of data, also makes it possible

The obtainment of images is performed by a scanner (ex Stratec XCT Research M instrument scanner (Norland Medical Systems, Fort.) specifically for the analysis of small grafting sites.

There have been many differences in bone tissue preparation techniques since the first tests in the 30s, yet the desire for the recovery of lost bone tissue among physicians and dentists still remains. The Ministry of Health has recently expressed interest in increasing and promoting this kind of transplant in the country. It is a fact that an increasing number of patients with bone loss for various reasons have approached specialized orthopedics and dentistry services in pursuit of reconstructive solutions. This context includes procedures

In dentistry, the officialization of allograft use arrives very late in 2005. Dental transplanters were curtailed in their use of this kind of treatment for more than a decade. Public policies of oral health were also very late in arriving. The result is perceptible in the statistics that evidence a high rate of edentulism in our population. (Ministerio da Saúde, 2003). In response to this situation, we can also observe high rates of prosthetic rehabilitations in the

Data from the Epidemiological Oral Health Survey in Brazil (2003) shows that about 67% of the population between 65 and 74 years of age use some kind of prosthesis as a form of edentulism treatment. We know that edentulism and the prolonged use of these prostheses lead to maxillary and mandibular bone resorption over the years (Davis, 1995; Gassen et al,

involving the use of allografts made available by musculoskeletal tissue banks.

process of bone grafts in patient to be submitted to future dental implants.

**14.3 Peripheral quantitative computed bone tomography (pQCT)** 

to evaluate the cortical and trabecular bones separately.

The parameters evaluated in this technique are:




**15. Discussion** 

Brazilian population.


proteins.


RESORPTION PARAMETER


The histomorphometric parameters adopted in this technique should follow a universal nomenclature agreed upon by the American Society of Bone and Mineral Research - ASBMR (Parfitt et al., 1987).

#### **14.2 Immunohistochemistry**

With the advent of osseointegrated dental implants it is extremely important for us to know the cellular processes involved in the osseointegration of allografts, as already mentioned, and this is possible with the immunohistochemistry technique. In vivo mineralization on the implant surface is directly related to the components of the extracellular bone matrix, both collagen and non-collagen components. The bone structure is composed of about 70% of inorganic matter (hydroxyapatite), while the rest is the organic matrix, predominantly consisting of collagen (95%), which is responsible for its flexibility and resistance whereas type I collagen provides support to the mineral structure. The non-collagen components include osteocalcin, osteonectin and osteopontin.

Osteocalcin is related to bone matrix mineralization, and is exclusively expressed by osteoblasts (Ducy et al. 1995). Osteonectin influences the synthesis of extracellular matrix components (Bradshaw et al. 2003) and the calcification of the organic matrix as it is selectively linked to hydroxyapatite and type I collagen fibrils (Termine et al. 1981). Osteopontin is strongly linked to hydroxyapatite crystals and is involved in the anchoring of the osteoclasts in the inorganic bone matrix, controlling crystal nucleation and growth.

Hence the evaluation of these proteins in the osseointegration process can provide us with important information about how the allogeneic bone graft used can corroborate for better interaction of the cellular mechanisms between donor and recipient focusing on the maintenance of the bone tissue after the implant installation.

Moreover, the remodeling of this matrix needs specialized enzymes. Matrix metalloproteinases (MMPs) are an important family of endopeptidases, with 25 known human members, and represent the largest class of enzymes that, collectively, are responsible for the degradation or resorption of extracellular matrix components, and are the only enzymes capable of cleaving fibrillar collagens (Curran et al. 1999), several proteins from the cellular surface and from the pericellular environment and some intracellular proteins.

This technique makes it possible to evaluate bone matrix proteins (type I collagen, osteocalcin, osteopontin and osteonectin) and the MMPs during the osseointegration process of bone grafts in patient to be submitted to future dental implants.

#### **14.3 Peripheral quantitative computed bone tomography (pQCT)**

Peripheral quantitative computed tomography - pQCT is a method used to measure the appendicular skeleton that, besides furnishing a broad range of data, also makes it possible to evaluate the cortical and trabecular bones separately.

The obtainment of images is performed by a scanner (ex Stratec XCT Research M instrument scanner (Norland Medical Systems, Fort.) specifically for the analysis of small grafting sites.

The parameters evaluated in this technique are:


60 Current Frontiers in Cryopreservation

Number of osteoblasts (**N.Ob**): absolute number of osteoblasts present in the measured

Number of osteoblasts by tissue area (**N.Ob/T.Ar**): number of osteoblasts by tissue area

Number of osteoblasts by bone perimeter (**N.Ob/B.Pm**): number of osteoblasts by bone

Osteoclast surface (**Oc.S/BS**%): percentage of trabecular surface that presents

Number of osteoclasts (**N.Oc**): number of osteoclasts present in the area of the bone

Resorption surface (**ES/BS** %): percentage of surface that presents bone resorption

The histomorphometric parameters adopted in this technique should follow a universal nomenclature agreed upon by the American Society of Bone and Mineral Research - ASBMR

With the advent of osseointegrated dental implants it is extremely important for us to know the cellular processes involved in the osseointegration of allografts, as already mentioned, and this is possible with the immunohistochemistry technique. In vivo mineralization on the implant surface is directly related to the components of the extracellular bone matrix, both collagen and non-collagen components. The bone structure is composed of about 70% of inorganic matter (hydroxyapatite), while the rest is the organic matrix, predominantly consisting of collagen (95%), which is responsible for its flexibility and resistance whereas type I collagen provides support to the mineral structure. The non-collagen components

Osteocalcin is related to bone matrix mineralization, and is exclusively expressed by osteoblasts (Ducy et al. 1995). Osteonectin influences the synthesis of extracellular matrix components (Bradshaw et al. 2003) and the calcification of the organic matrix as it is selectively linked to hydroxyapatite and type I collagen fibrils (Termine et al. 1981). Osteopontin is strongly linked to hydroxyapatite crystals and is involved in the anchoring of the osteoclasts in the inorganic bone matrix, controlling crystal nucleation and growth.

Hence the evaluation of these proteins in the osseointegration process can provide us with important information about how the allogeneic bone graft used can corroborate for better interaction of the cellular mechanisms between donor and recipient focusing on the

Moreover, the remodeling of this matrix needs specialized enzymes. Matrix metalloproteinases (MMPs) are an important family of endopeptidases, with 25 known human members, and represent the largest class of enzymes that, collectively, are responsible for the degradation or resorption of extracellular matrix components, and are the only enzymes capable of cleaving fibrillar collagens (Curran et al. 1999), several proteins

area;

RESORPTION PARAMETER

osteoclasts;

(Parfitt et al., 1987).

**14.2 Immunohistochemistry** 

tissue evaluated;

analyzed, expressed in square millimeters;

perimeter analyzed, expressed in millimeters.

lacunae with or without the presence of osteoclasts.

include osteocalcin, osteonectin and osteopontin.

maintenance of the bone tissue after the implant installation.


### **15. Discussion**

There have been many differences in bone tissue preparation techniques since the first tests in the 30s, yet the desire for the recovery of lost bone tissue among physicians and dentists still remains. The Ministry of Health has recently expressed interest in increasing and promoting this kind of transplant in the country. It is a fact that an increasing number of patients with bone loss for various reasons have approached specialized orthopedics and dentistry services in pursuit of reconstructive solutions. This context includes procedures involving the use of allografts made available by musculoskeletal tissue banks.

In dentistry, the officialization of allograft use arrives very late in 2005. Dental transplanters were curtailed in their use of this kind of treatment for more than a decade. Public policies of oral health were also very late in arriving. The result is perceptible in the statistics that evidence a high rate of edentulism in our population. (Ministerio da Saúde, 2003). In response to this situation, we can also observe high rates of prosthetic rehabilitations in the Brazilian population.

Data from the Epidemiological Oral Health Survey in Brazil (2003) shows that about 67% of the population between 65 and 74 years of age use some kind of prosthesis as a form of edentulism treatment. We know that edentulism and the prolonged use of these prostheses lead to maxillary and mandibular bone resorption over the years (Davis, 1995; Gassen et al,

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 63

In the next few years we will observe growth of the elderly population in our country (around 13% in 2020), motivated by the increase in life expectancy. It is estimated that in 2020 this expectancy will be 71.2 years for men and 74.7 years for women (IBGE, 2011). Considering that the elderly population is exposed to factors accumulated during their lifetimes that lead to the need for pre-prosthetic reconstructive bone interventions (edentulism, prolonged use of prostheses, osteoporosis, hypothyroidism) we can reflect here on a possible influence over prosthetic rehabilitations of the elderly population in future years, and that in some of them the use of biomaterials (including allograft) may be

What then has become an obvious trend gives rise to the need to discuss the future of dental transplants. Public health surveillance policies seek, together with trade associations (Regional Federal Council of Dentistry) and tissue banks, the assurance of traceability in procedures of this nature. This is undoubtedly the major challenge for tissue banks. Unlike other kinds of transplants, where the data are compiled and made available by hospitals, in dental transplantations there is the need for total involvement of the transplanting professional in disclosing and informing the adverse effects detected in the treatment. Following the example of the national policy implemented by ANVISA in the area of hemoderivates transfusion, the intention is to create a similar system for the surveillance of tissue transplants in the country, which will include dental transplants. In this model, the adverse effects can be notified nationwide (electronically), enabling the construction of a database that will serve as a source for the definition of corrective and preventive actions of complications in dental surgeries with the use of allografts. Such information is extremely

valuable to banks, particularly in the quality control of the tissues produced thereby.

blocks, especially for use in sinus lifting surgery preceding implant installation.

After a more detailed analysis of dental transplants, we detected that approximately 76% are related to the use of cortical tissues, which in our understanding is expected due to the greater availability of this type of tissue at banks. While a spongy segment of a long bone such as the femur and tibia (metaphyseal region) yields 10 to 15 units of spongy blocks, 50 to 60 units of cortical blocks are processed from a diaphysis. It should also be considered that due to the lesser availability of spongy tissues, surgeons have opted in particular for cortical

**GRAFT MORPHOLOGY Relative Frequency (%)**  Cortical 75.64 % Spongy 15.86 % Cortico-Spongy 8.5 % **TOTAL 100.00**  Table 3. Distribution of Grafts in Terms of Morphology. BTM (musculoskeletal tissue bank) -

The most frequent surgical site was undoubtedly the maxilla (98.2%- Graph 4) and we hereby emphasize the main reasons for this finding. Firstly, edentulism in the Brazilian population has appeared to affect the maxilla more than the mandible which certainly leads to the greater use of upper prostheses (57.9%) versus lower prostheses (34.18%) by the population aged between 65 and 74 years (Ministério da Saúde, 2003). Secondly, the actual anatomy of the maxilla due to the presence of maxillary sinuses that are grafted during the

indicated.

HCFMUSP, 2006 to 2010.

2008). The high rate of individuals with this necessity, the greater economic access of the population to treatments with osseointegrated implants and to the start of authorization of allograft use in dentistry, corroborated the abrupt growth of bone transplants in dentistry at around **600%** in these last 5 years (**Graph 2**).

Graph 2. Annual Distribution of homologous tissues made available by a tissue bank that serves as a national reference for Dental Transplants (2006 to 2010).

Such a situation was also observed in our study in relation to the number of transplanting dentists, whose number climbed steeply from **22** to **3585** also in 5 years (**Graph 3**).

Graph 3. Absolute Frequency of dentists accredited for transplants over the years (2006- 2010)

2008). The high rate of individuals with this necessity, the greater economic access of the population to treatments with osseointegrated implants and to the start of authorization of allograft use in dentistry, corroborated the abrupt growth of bone transplants in dentistry at

Graph 2. Annual Distribution of homologous tissues made available by a tissue bank that

dentists, whose number climbed steeply from **22** to **3585** also in 5 years (**Graph 3**).

Graph 3. Absolute Frequency of dentists accredited for transplants over the years (2006-

2010)

Such a situation was also observed in our study in relation to the number of transplanting

serves as a national reference for Dental Transplants (2006 to 2010).

around **600%** in these last 5 years (**Graph 2**).

In the next few years we will observe growth of the elderly population in our country (around 13% in 2020), motivated by the increase in life expectancy. It is estimated that in 2020 this expectancy will be 71.2 years for men and 74.7 years for women (IBGE, 2011). Considering that the elderly population is exposed to factors accumulated during their lifetimes that lead to the need for pre-prosthetic reconstructive bone interventions (edentulism, prolonged use of prostheses, osteoporosis, hypothyroidism) we can reflect here on a possible influence over prosthetic rehabilitations of the elderly population in future years, and that in some of them the use of biomaterials (including allograft) may be indicated.

What then has become an obvious trend gives rise to the need to discuss the future of dental transplants. Public health surveillance policies seek, together with trade associations (Regional Federal Council of Dentistry) and tissue banks, the assurance of traceability in procedures of this nature. This is undoubtedly the major challenge for tissue banks. Unlike other kinds of transplants, where the data are compiled and made available by hospitals, in dental transplantations there is the need for total involvement of the transplanting professional in disclosing and informing the adverse effects detected in the treatment. Following the example of the national policy implemented by ANVISA in the area of hemoderivates transfusion, the intention is to create a similar system for the surveillance of tissue transplants in the country, which will include dental transplants. In this model, the adverse effects can be notified nationwide (electronically), enabling the construction of a database that will serve as a source for the definition of corrective and preventive actions of complications in dental surgeries with the use of allografts. Such information is extremely valuable to banks, particularly in the quality control of the tissues produced thereby.

After a more detailed analysis of dental transplants, we detected that approximately 76% are related to the use of cortical tissues, which in our understanding is expected due to the greater availability of this type of tissue at banks. While a spongy segment of a long bone such as the femur and tibia (metaphyseal region) yields 10 to 15 units of spongy blocks, 50 to 60 units of cortical blocks are processed from a diaphysis. It should also be considered that due to the lesser availability of spongy tissues, surgeons have opted in particular for cortical blocks, especially for use in sinus lifting surgery preceding implant installation.


Table 3. Distribution of Grafts in Terms of Morphology. BTM (musculoskeletal tissue bank) - HCFMUSP, 2006 to 2010.

The most frequent surgical site was undoubtedly the maxilla (98.2%- Graph 4) and we hereby emphasize the main reasons for this finding. Firstly, edentulism in the Brazilian population has appeared to affect the maxilla more than the mandible which certainly leads to the greater use of upper prostheses (57.9%) versus lower prostheses (34.18%) by the population aged between 65 and 74 years (Ministério da Saúde, 2003). Secondly, the actual anatomy of the maxilla due to the presence of maxillary sinuses that are grafted during the

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 65

grafting period. Data of extreme importance, in view of the need for assurance of

And finally, immunohistochemistry solves some mysteries related to the cellular processes involved in osseointegration. The technique allows us to evaluate, by means of reaction by selected antibodies, the expression of proteins related to the mineralization process

As the basis for all these cellular events, the use of specific antibodies (Tartrate-resistant Acid Phosphatase - TRAP, Osteoprotegerin, Rank-L and Cd34) also allows us to evaluate the inflammation and the vascularization of grafting areas throughout the osseointegration period. Nowadays in Brazil we are experiencing a time of transition in the indication of these transplants, and studies with this information content are certainly necessary for better knowledge and indication of allografts in dentistry, as well as the criteria and

[1] Aaron JE, Shore PA. Bone histomorphometry: concepts and commom techniques. In: An

[2] Amatuzzi MM, Croci AT, Giovani AMM, Santos LAU, Maragni GG, Shinzato JC. Banco

[3] Amatuzzi MM, Croci AT, Giovani AMM, Santos LAU. Banco de tecidos:estruturação e

[4] American Association of Tissue Banks. Standards for Tissue Banking. 11th ed. McLean :

[5] Aubin JE, Lian JB, Stein GS. Bone formation: maturation and functional activities of

[6] Bancroft JD, Stevens A. Theory and practice of histological techniques.Fouth Edition.

[7] Barros Filho TEP, Rossi JDMBA, Rodrigues CJ, Lage LA, Reis PR. Poder osteogênico dos

homólogo irradiado e homólogo AAA. *Rev Bras Ortop*. 1989; 24(1-2):36-40. [8] Bitar AC, Santos LAU, Croci AT, Pereira JARM, França Bisneto EN, Giovani AMM,

[9] Boldt JG, Dilawari P, Agarwal S, Drabu KJ. Revision total hip arthroplasty using

[10] Bradshaw, A.D., Graves, D.C., Motamed, K., & Sage, E.H. 2003. SPARC-null mice

*Proc.Natl.Acad.Sci.U.S.A*, 100, (10) 6045-6050 available from: PM:12721366 [11] Brasil, Leis etc. Decreto n.2268 de 30 de junho de 1997. Dispõe sobre a remoção de

*Diário Oficial da União*, Brasília (DF). 1997 30 jun; seção 1:1.

YH, Martin KL. Handbook of histology methods for bone and cartilage. New

de tecidos:estruturação e normatização. In: Amatuzzi, Joelho:articulação dos

osteoblast lineage cells. In: American Society for Bone and Mineral Research. Primer on the metabolic bone diseases and disorders of mineral metabolism. 6th ed.

enxertos ósseos: estudo experimental comparativo entre enxertos autólogo,

Oliveira CRGCM. Histological Study of Fresh versus Frozen Semitendinous Muscle

impaction bone grafting with cemented nonpolished stems and charnley cups. *J* 

exhibit increased adiposity without significant differences in overall body weight .

órgãos, tecidos e partes do corpo humano para fins de transplantes e tratamento.

mechanical stability of dental implants, when implanted in the newly formed bone.

(osteocalcin, collagen) and remodeling of bone grafts (osteopontin, osteonectin).

methodologies that can be used to analyze osseointegration.

Jersey: Humana Press; 2003. p. 331-51.

membros inferiores. São Paulo:Roca; 2004. p.687-99.

normatização. *Rev Bras Ortop.* 2000; 35(5):165-72.

American Association of Tissue Banks; 2007.

Churchill Livingstone. United Kingdom, 1999.

Tendon Allografts. CLINICS 2010;65(3):297-303

Washington: ASBMR; 2006. p.20-9.

*Arthroplasty*. 2001; 16(8):943-52.

**16. References** 

sinus lifting technique. This technique is very frequent in the branch in implant dentistry. Thirdly, the lower bone quality of the maxilla in comparison to the mandible, focusing on the primary stability of implants and propensity for resorption after the prolonged use of prostheses. (Mezzomo et al, 2010) It is also worth emphasizing that the maxilla appears as a more esthetic area that demands more attention from individuals concerned about its rehabilitation.

Graph 4. Distribution of Type and Surgical Site of use of the tissues made available by a bank that serves as a national reference from 2006 to 2010.

Although epidemiological data shows the increasing use of homologous tissues in dental surgery, few studies in our field assess their efficacy, hence the need to establish consensuses on their applicability and more importantly, predictability of treatments with the use of allografts. We take predictability to mean knowledge of the responses of bone tissue to the interventions performed in our areas of specialty. Thus in this study we have highlighted a chapter on possible lines of investigation enforceable in our field, which can help us in our search for answers related to the osseointegration process of allografts at sites of dental interest.

The analysis of grafting area by histomorphometry allows us to accurately quantify the structure of the grafted tissue (area, volume) and how much tissue was formed after interaction with the recipient's body (osteoid volume, osteoid surface), to judge the participation of each bone cell (number of osteoblasts and osteocytes) and also to quantify resorptions that have occurred (resorption surface) as a result of this interaction. This data is supplemented by peripheral quantitative computed tomography – pQCT, which unlike histomorphometry, allows us to evaluate the density of the tissue before and after the grafting period. Data of extreme importance, in view of the need for assurance of mechanical stability of dental implants, when implanted in the newly formed bone.

And finally, immunohistochemistry solves some mysteries related to the cellular processes involved in osseointegration. The technique allows us to evaluate, by means of reaction by selected antibodies, the expression of proteins related to the mineralization process (osteocalcin, collagen) and remodeling of bone grafts (osteopontin, osteonectin).

As the basis for all these cellular events, the use of specific antibodies (Tartrate-resistant Acid Phosphatase - TRAP, Osteoprotegerin, Rank-L and Cd34) also allows us to evaluate the inflammation and the vascularization of grafting areas throughout the osseointegration period.

Nowadays in Brazil we are experiencing a time of transition in the indication of these transplants, and studies with this information content are certainly necessary for better knowledge and indication of allografts in dentistry, as well as the criteria and methodologies that can be used to analyze osseointegration.

### **16. References**

64 Current Frontiers in Cryopreservation

sinus lifting technique. This technique is very frequent in the branch in implant dentistry. Thirdly, the lower bone quality of the maxilla in comparison to the mandible, focusing on the primary stability of implants and propensity for resorption after the prolonged use of prostheses. (Mezzomo et al, 2010) It is also worth emphasizing that the maxilla appears as a more esthetic area that demands more attention from individuals concerned about its

Graph 4. Distribution of Type and Surgical Site of use of the tissues made available by a

Although epidemiological data shows the increasing use of homologous tissues in dental surgery, few studies in our field assess their efficacy, hence the need to establish consensuses on their applicability and more importantly, predictability of treatments with the use of allografts. We take predictability to mean knowledge of the responses of bone tissue to the interventions performed in our areas of specialty. Thus in this study we have highlighted a chapter on possible lines of investigation enforceable in our field, which can help us in our search for answers related

The analysis of grafting area by histomorphometry allows us to accurately quantify the structure of the grafted tissue (area, volume) and how much tissue was formed after interaction with the recipient's body (osteoid volume, osteoid surface), to judge the participation of each bone cell (number of osteoblasts and osteocytes) and also to quantify resorptions that have occurred (resorption surface) as a result of this interaction. This data is supplemented by peripheral quantitative computed tomography – pQCT, which unlike histomorphometry, allows us to evaluate the density of the tissue before and after the

bank that serves as a national reference from 2006 to 2010.

to the osseointegration process of allografts at sites of dental interest.

rehabilitation.


Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives 67

[31] Giannoudis PV, Dinapoulos H, Tsiridis E. Bone substitutes: an update. Injury. 2005;

[32] Giovani AMM, Croci AT, Oliveira CRGCM, Filippi RZ, Santos LAU, Maragni GG.

[33]Giovani AMM. *Estudo comparativo entre o tecido ósseo criopreservado e o conservado em glicerol a* 

[34] Groves EWH. Methods and results of transplantation of bone in the repair of defects

[35] Heyligers IC, Klein-Nulend J. Detection so living cells in non-processed but deep-frozen

[36] IBGE: Instituto Brasilerio de Geografia e Estatística: http://www.ibge.gov.br/home/ .

[37] Janssen ME, Lam C, Beckham R. Outcomes of allogenic cages in anterior and posterior

[38] Jorgetti V. Biópsia óssea e análise histomorfométrica. Manual Fleury de diagnóstico de

[39] Junqueira LC, Carneiro J. Histologia básica. 9a ed. Rio de Janeiro :Guanabara-Koogan;

[40] Knighton DR, Hunt TK, Thakral KK, Goodson WH 3rd. Role of platelets and fibrin in

[41] Lavernia CJ, Malinin TI, Temple HT, Moreyra CE. Bone and tissue allograft use by

[42] Lindhe J, Karring T, Lang NP.Tratamento periodontal regenerativo. Tratado de

[43] Malafaya, PB, G. A. Silva, E. T. Baran, R. L. Reis, Curr.Opin. Solid State Mater. Sci. 2002,

[44] Mecall RA, Rosenfeld AL. The influence of residual ridge resorption patterns on

[45] Mezzomo RJ, Garbin CA, Schuh C, Rigo L. Critical analysis of studies of immediately

[46] Ministério da Saúde : Levantamento das condições de saúde bucal da população brasileira; 2003. Disponível em : http://dab.saude.gov.br/CNSB/vigilancia.php [47] Moore KL. Anatomia Orientada para a Clínica. 2ª ed. Rio de Janeiro - RJ: Editora

[48] Nather A. Organisation, operational aspects and clinical experience of National University of Singapore Bone B. *Ann Acad Med Singapore*. 1991; 20(4):453-7. [49] Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone

the healing sequence: an in vivo study of angiogenesis and collagen synthesis. *Ann* 

periodontia clínica e implantologia oral. 3a ed. Rio de Janeiro: Guanabara

implant fixture placement and tooth position. Part I. Int J Periodontics Restorative

loaded implants supporting fixed prostheses in the edentulous maxxila.

histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. *J Bone Miner Res.* 1987;

ASBMR; 2006. p.42-48.

glycerol solution. Clinics. 2006;61(6):565-70.

bone allografts. *Cell Tissue Bank.* 2005; 6 :25-31.

doenças ósteo-metabólicas. 2003. Disponível em:

Acessado em 15 de julho de 2011.

1999. Cap.8, p.111-28: Tecido ósseo.

*Surg*. 1982; 196 (4): 379-88.

Koogan;1997. p.428-62.

Dent. 1991; 11(1): 8-23

Implantnews 7 (4), 2010.

Guanabara. 1990.

2(6):595-610

6, 283.

caused by injury or disease. *Br J Surg*. 1917; 5(18):185-242.

lumbar interbody fusion. *Eur Spine J*. 2001; 10(Suppl 2):158-68.

http://www.fleury.com.br/htmls/cdrom/capitulo5.htm.

orthopaedic surgeons. *J Arthroplasty.* 2004; 19(4):430-35.

36S:S20-27.

metabolic bone diseases and disorders of mineral metabolism. 6th ed. Washington:

Comparative study of cryopreservedbone tissue and tissue preserved in a 98%

*98%* [dissertação]. São Paulo: Faculdade de Medicina, Universidade de São Paulo; 2005.


[12] Brasil, Leis etc. Lei n.9434 de 5 de fevereiro de 1997. Dispõe sobre a remoção de órgãos,

[13] Brasil, Leis etc. Portaria n.1686 de 20 de setembro de 2002. Dispõe sobre a

[14] Brasil, Leis etc. PORTARIA Nº 2.600, DE 21 DE OUTUBRO DE 2009. Aprova o

[15] Brasil, Leis etc. Resolução n. 220 de 27 de dezembro de 2006. Dispõe sobre o

[16] Cabrita, H.B., et al., Prospective study of the treatment of infected hip arthroplasties

[17] Carrel A. The preservation of tissues and its applications in surgery. 1912. *Clin Orthop* 

[18] Contar CM, Sarot JR, da Costa MB, Bordini J, de Lima AA, Alanis LR, Trevilatto PC,

[19] Croci AT, Camargo OP, Oliveira CRGC, Baptista AD, Sorrilha A. Estudo histológico dos enxertos ósseos homólogos humanos. *Acta Ortop Bras.* 2003b; 11(4):220-4. [20] Cunningham N, Reddi AH. Biologic principles of bone induction: application to bone

[21] Curran, S. & Murray, G.I. 1999. Matrix metalloproteinases in tumour invasion and

[22] Drumond SN. Transplantes ósseos. In: Pereira WA. *Manual de transplantes de órgãos e* 

[23] Ducy, P. & Karsenty, G. 1995. Two distinct osteoblast-specific cis-acting elements

[24] Enneking WF, Burchardt H, Puhl JL, Piotrowski G. Physical and biological aspects of repair in dog cortical-bone transplants. *J Bone Joint Surg Am.* 1975; 57:237-52. [25] European Association of Tissue Banks. Common Standards for Tissues and Cells

[26] Fischer LP, Fischer-Athiel C, Fischer BS. One hundred years of bone surgery in the Lyons Teaching Hospitals (1897-1997). *Ann Chir*. 1998; 52(3):264-78. [27] Fonseca RJ, Davis WH .Reconstructive Preprosthetic Oral and Maxillofacial Surgery, ed

[28] Galea G, Kearney. Clinical effectiveness of processed and unprocessed bone. *Transfus* 

[29] Gassen, Humberto Thomazi; Filho, Rolf Muner; Siqueira, Bianca Munari de; Oliveira,

utilizando enxerto de ramo mandibular. Stomatos, v.14, n.26, jan./jun. 2008. [30] Gerstenfeld LC, Einhorn TA. Fracture healing: the biology of bone repair and

Samia Bohm; Junior, Aurelício Novaes Silva. Reconstrução óssea de maxila atrófica

regeneration. In: American Society for Bone and Mineral Research. Primer on the

metastasis. *J.Pathol.*, 189, (3) 300-308 available from: PM:10547590

Banking: Berlin: European Association of Tissue Banks; 2004.

*tecidos*. 2a ed. Rio de Janeiro: Medsi; 2000. p.359-80.

2. Philadelphia, WB Saunders, 1995, pp 498-510

*Oficial da União*, Brasília (DF). 1997 5 fev; seção 1:25.

Brasília (DF). 2009 21out.

Brasília (DF). 2006 29 dez.

2007. 62(2): p. 99-108.

*Relat Res.* 1992; 278:2-8

W.B . Saunders C; 1992. p.93-8.

available from: PM:7891679

*Med*. 2005; 15(3):165-74.

*Diário Oficial da União*, Brasília (DF). 2002 24 jul; seção 1:1.

histologic analysis. J Oral Implantol. 2011 Apr;37(2):223-31.

tecidos e partes do corpo humano para fins de transplantes e tratamento. *Diário* 

regulamentação para funcionamento de banco de tecidos músculo esqueléticos.

Regulamento Técnico do Sistema Nacional de Transplantes. *Diário Oficial da União*,

Regulamento Técnico para o Funcionamento de Bancos de Tecidos Musculoesqueléticos e de Bancos de Pele de origem humana. *Diário Oficial da União*,

with or without the use of an antibiotic-loaded cement spacer. Clinics (Sao Paulo),

Machado MÂ. Fresh-frozen bone allografts in maxillary ridge augmentation:

grafts. In: Habal MB, Reddi AH. Bone grafts and bone substitutes.Philadelphia:

control expression of a mouse osteocalcin gene *Mol.Cell Biol.*, 15, (4) 1858-1869

metabolic bone diseases and disorders of mineral metabolism. 6th ed. Washington: ASBMR; 2006. p.42-48.

	- http://www.fleury.com.br/htmls/cdrom/capitulo5.htm.

**4** 

**Validation of Primary Packaging for** 

**Cryopreserved Musculoskeletal Tissues** 

\*Luiz Augusto Ubirajara Santos1, Rosa Maria Vercelino Alves2, Alberto T. Croci1, Fábio Gomes Teixeira2, Paulo Henrique Kiyataka2, Marisa Padula2, Mary Ângela Fávaro Perez2, Monica Beatriz Mathor3, Renata Miranda Parca6, Arlete M.M . Giovani1, Cesar Augusto Martins Pereira4, Graziela Guidoni Maragni1, Thais Queiróz Santolin1 and Lucas da Silva Pereira5

Bones and tendons are obtained from donors who have been pronounced brain dead after a rigid and extensive screening process. The tissues obtained are sent to a Tissue Bank and submitted to processing steps, packaging and cryopreservation at -80◦C. It is vital to maintain sterility and integrity, so that no tissue is discarded.These precautions also extend to the packaging, which should promote containment and protection. Although minimum technical criteria have already been defined for the food industry, this has still not been regularized in Brazil. We emphasize, therefore, the need to study this subject, focusing on maintaining the quality of the musculoskeletal tissues produced by tissue banks. All procedures developed byTissue Banks currently present in the country have rigid control over the quality and the traceability of tissues made available, based on international standards (EATB, 2004; AATB, 2007) on the legislation (BRASIL, 1997-2006) and in

In the cryopreservation room, the tissues are stored according to their status in the process. Thus, there are designated areas for tissues under analysis or in quarantine (where they remain for around 60 days before the result of all the exams) and for those that have been liberated for use. both areas are equipped with ultra-low temperature freezers, with

*1Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of São Paulo School of* 

*2Packaging Technology Center – Institute of Food Technology, Campinas, SP, Brazil 3Nuclear and Energy Research Institute - IPEN/CNEN-SP – São Paulo, Brazil 4Biomechanics Laboratory - Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of* 

*5Dental Student Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of São Paulo* 

conformity with Good Manufacturing Practices - GMP.

**2. Cryopreservation of musculoskeletal tissues** 

temperatures ranging from minus 80 to minus 100 degrees Celsius.

**1. Introduction** 

*Medicine, Sao Paulo, SP, Brazil* 

*São Paulo school of Medicine, Brazil* 

*school of Medicine, São Paulo, SP, Brazil 6National Health Surveillance Agency- ANVISA*\*

	- http://www.abto.org.br/profissionais/profissionais.asp.

http://www.teses.usp.br/teses/disponiveis/5/5140/tde-16082007-160750/

