Bone grafts and substitutes
in Orthopaedic surgery

G.A. MAXERAS, P. MEGAS, G. MPAMPIS, E. THOMAS, E. VAVOURAKI*
* This study was performed following an order from KESY decision No ÄÕ1ä/Ã.Ð. 18292 concerning the indication's and limitation's determination for grafts use in orthopaedic diseases.

Mailing address:
G.A. Macheras
18 Papadiamantopoulou St.
115 28 Athens

INTRODUCTION
Bone grafts usage frequency is increasing day by day in several orthopaedic surgery operations. In USA, according to American Academy of Orthopaedic Surgeons 2002 data (Dallas Texas) it is estimated that more than 500.000 bone graft operations are performed annually. Almost half of them concern spinal fusion surgeries. There are no data in Greece concerning the precise number of these bone graft transplantations, but there is a general impression that their number is greatly increasing, as well as the amount of bone graft used is also increasing.
Whilst 3-4 years ago the only bone graft source (autograft) was patient itself and to a lesser degree allografts were used either from Democritus Center or from bone grafts banks located in some hospitals (mainly femoral head and condyles), now days a vast consumption of industrialized bone grafts is noticed.
According to IKA data the usage of these grafts shows a huge increase without any clear indications for their utilization necessity, the appropriate graftÕs type but also the result of its usage.
Bone grafts and substitute biology is assessed from the idea of bone production, including osteogenesis, osteoinduction and osteoconduction. All bone grafts and their substitutes must and can be assessed through the above procedures. There is no doubt that recent (fresh) spongy and to a smaller extent cortical autograft are fulfilling all of the above properties.
There is no risk for disease transmission and they present excellent behavior, providing a satisfying stability and having perfect mechanical properties after their integration. Thus, they make the gold standard for other allografts and their synthetic substitutes. However, the available autograft quantities are limited and at the same time their administration is combined with a surgical time increase, blood losing, postoperative pain and potential hospitalization period extension[1,21].
The above evidence's recognition led orthopaedic surgeons to search for alternative solutions, consisting of allografts and bone grafts substitutes. Bone grafts that can be used are those based upon natural origin materials (i.e. demineralized human bone matrix, bovine collagen mineral composites, growth factors and processed coralline hydroxyapatite) and synthetic materials (i.e. calcium sulfate pellets, bioactive glass and calcium phosphate cement).
Some of the above have osteoinductive properties and some other present with osteoconductive ones. The basic query concerning these grafts is whether and in what extent they aid fractureÕs porosis, osseous defects filling, spinal fusion, e.t.c. and if their use is really necessary, when and in what amount they should be utilized. Today, there are no published studies offering important information because they are all supported by graft producing companies and therefore, they are not objective and they mainly refer to case reports graft use and not to large randomized trials. Furthermore, there are no prospective double combining studies between patients receiving autografts and patients receiving bone substitutes (grafts), in order to confirm their action.
Additionally, there is a great concern about the likelihood of transmitting contagious diseases with these products despite the fact that transmission risk seems to be very low. European Union is looking forward to establishing definite indications and usage conditions for these products.
Finally, substituteÕs cost is an important factor. For that reason studies proving that their implementation can evidently decrease pseudoarthrosis and fracture's porosis time are needed. An attempt to determine the indications and limitations of bone grafts use is made after taking into consideration all these concerns, as well as the international standards and after studying the limited international references.

GENERAL INFORMATION
Tissues transplantations promote and improve life quality of million people per year globally and of many thousands in Greece. Bone is the commonest transplanted human tissue. Increasing bone grafts needs were met by using processed grafts of human origin, in Greek and European level, at late 1960s. Simultaneously, processed animal grafts are circulating, mainly bovine, as well as synthetic ones (from inorganic or organic raw materials).
Bone transplantation plays an increasing role in restorative orthopaedic surgery due to the more complex reparation techniques of large osseous defects applied today. Fractures and their complications (pseudoarthrosis)61 treatment, benign and malignant tumors and congenital bone disorders restoration are all fields for its implementation. Additionally, arthroplasties indications expansion to joints with defect bone infrastructure and moreover, the increasing rate of their revision are often requiring the use of grafts[3,22]. Bone transplantation basic goals are either the biological reinforcement of bone production (pseudoarthrosis, joint fusions), or the skeleton structural integrity restoration (tumors, arthroplasties), or both. The increasing demand resulted in the presentation of many graft types.
There are virtually no laws concerning production and distribution of bone grafts in Greece. Laws dealing with tissue and organ transplantations are 1383/83 and 2737/99, which posed the principles of tissue and organ removing and donating from live or dead donors. Lately, Central Health Board (KESY) and National Transplantation Organization (EOM) created the foundation and operation principles of Tissue Banks in Greece. With respect to bone graft circulation and distribution, the following principles are applied:
Processed animal bone grafts packs come into circulation after Greek Drugs Administration (EOF) and they come under the E.U Directive 42/93, as it is implemented into Greek Legislation by Ministerial Decision ÄÕ7/2480/94.
In Europe a definite product demarcation exists.
European Union Directive 42/93 for "medical technological products" includes animal bone grafts and regulates their production and circulation rules. At present there is no specific direction for bone grafts of human origin either they are derived from specially processed tissues or from cryopreserved ones. With this goal in mind, European Association of Tissue Banks (EATB) as well as European Association of MusculoSkeletal Tissues (EAMST) are taking action and exerting pressure, while they processed and edited guidelines for Tissue Bank operation, in order to establish uniform General Standards for grafts quality.
These guidelines cover technical and ethical issues related to tissue collection and process, as well as to graft production and distribution. Production guidelines are based on ISO 9001 series philosophy and in some EU member countries they are already part of National Legislation.
In France:
Cryopreserved tissues supply occurs in hospitals where Banks are operating, whereas processed bone grafts supply takes place commercially. Prices of products are determined by the Commission responsible for their circulation. Public Hospitals make their bone grafts orders from Banks or Trade according to their approved budget. Private Hospitals obtain bone grafts in a similar way for their patients.
Unfortunately, despite the repeating efforts for producing a European Union Directive concerning the bone graft production and circulation circumstances, in order to ensure products quality, security and effectiveness, only one preliminary plan has been produced and discussed (Porto 2000/6, Malaga 2000/2), but it has not been formed yet into a final Directive.
In Greece:
"Demokritus" Tissue Grafts Bank collects human origin tissues, manipulates them and produces grafts for medical purposes. It works according to international relevant standards and qualifications of the International Organization of Atomic Energy, of World Health Organization and EATB and EAMST, while being a member of them. Produced grafts are disposed free until now and they are ready for use in any hospital, clinic and medical laboratory of the country.
Available Bank grafts are including the following:
a) Bone (spongy, cortical, combined, skull parts), b) Dura Mater c) Fetal membranes d) Epidermis, e) Cartilage, f) Tendons, g) Nerves. The above grafts are available in different shapes and size.

BONE GRAFTS TYPES
Autografts are bone parts transplanted from one part to another in the same organism (i.e from iliac bone to tibia, e.t.c)[69,75]. Allografts come from a different organism of the same species (from human to human), whereas xenografts come from different organisms of different species (from cow to human). Bone substitutes are synthetic materials mimicking one or more bone grafts functions[13,29,31,43]. Bone grafts can be fresh, frozen, lyophilized or according to their shape spongy, cortical, corticospongy, and osteoarticular. Vascular autografts are those having vascular stems osculated with recipient area vessels during the operation.
Grafts biological potential is the sum of their bone production ability, osteoinductive and osteoconductive effect[4,38,78]. Osteogenesis (bone production) is new bone synthesis from surviving precursor osteoblasts and autograft osteoblasts[16,24,38,69,78]. Osteoinductive is new bone formation after the activation of multivalent mesenchymal host cells and their differentiation in chondroblasts and osteoblasts, mainly produced by graft factors generally named morphogenetic proteins[31,36,45,48,62,63,71,72,74]. Osteoconductive is the property in which graft acts like an inactive scaffold which is initially infiltrated by newly formed vessels transferring recipient precursor osteocytes forms, which diversify and mature resulting in a new bone.
Autografts are biologically more active than any other graft type because they are consisted of live osteocytes. Despite their definitely greater bone production potential, they show severe disadvantages like the inability of receiving large quantities and the significant mortality rate of the donor region[12,33,80]. Donor site local complications can be inflammation, hematoma, severe pain, lack of sensibility or deformed scar and hospitalization time extension. On the other hand, allografts are available in large quantities, do not present any donor local complications and their implementation results in a significant decrease in blood loss, operation time and total hospitalization period[2,19,25]. The most important allograft disadvantages are including the likelihood of transmitting contagious diseases and more specifically AIDS[9], their antigenicity and rejection, even in a much lesser degree than other tissues6 and their decreased biological effect after sterilization procedures[42]. Furthermore, their smaller bone production potential, their greater absorption rate and their smaller revascularization, compared with autografts, are well documented[15,16]. They also require complicated and expensive sterilization and storage procedures in graft banks.
Allografts are available as fresh, frozen or lyophilized (freeze-dried). Fresh allografts are no longer in use because they present many problems like small available time between their receipt and placement, production of immunological reaction and mainly because it is not possible to check for any potential donor disease (HIV- Hep-B,C) and their sterilization status.
Frozen allografts are preserved in deep freezers in -70°C. Freezing decreases their antigenicity without altering their genetic engineering properties. Their usage is very safe because enough time is provided in order to check for any potential contagious diseases. Lyophilization is the procedure of water removal (dehydration) from a frozen allograft. The product is placed in vacuum packing and preserved in room temperature for 5 years. This procedure also decreases grafts antigenicity, but the already decreased one induction property is not influenced and their genetic engineering properties are affected (less compression tolerance) especially after their rehydration.

MECHANISMS OF BONE GRAFTS INTEGRATION
Cortical grafts are integrated with a different mechanism than spongy ones. A slow graft's absorption is observed by osteoclasts absorbing haversian systems resulting in mechanical tolerance reduction. Osteoblasts appearance follows, thus producing and deposing new bone. Thereafter, new bone remodelling is taking place based on the exerted forces. In spongy grafts, due to the presence of empty spaces, newly formed vessels infiltration proceeds rapidly. Differentiation of mesenchymal non-differentiated cells into osteoblasts is following, which are positioned in old osseous trabeculae producing new bone. Grafts trabecula are then replaced with the procedure of procumbent substitution.

BONE GRAFT SUBSTITUTES
Due to the above mentioned problems presented by both autografts and allografts, researchers are switching to bone graft substitutes production. Bone substitutes are either natural or synthetic materials or derived from human or animal or other tissues processing and they are looking forward to restoring the structural and functional bone integrity in several kinds of bone defects or spinal fusions.
There are many types of bone graft substitutes which differ in synthesis, mechanical properties, osteoinductive and osteoconductive effects, but also in the way they are absorbed or reconstructed inside the human organism. The ideal bone graft substitute is the one being biocompatible, bioabsorbed in a controlled way, having osteoinductive and osteoconductive properties and showing similar structural resemblance with human bone, but at the same time it must be easily implanted and be quite cost saver[18].
Currently available bone graft substitutes can be divided in the following categories:
1. Demineralized Allograft Bone Matrix
2. Pottery materials
3. Mixtures of collagen, hydroxyapatite and calcium salts
4. Hydroxyapatite coming from corals
5. Calcium sulfate
6. Bioglasses
7. Processed bovine xenografts
While all of the above show osteoconductive properties[13,16,18,41,44,4951,58,67], however, osteoinductive properties are seen primary in first category grafts, that is to say, those having demineralized bone matrix (DBM)[4,21,39,60,71,76,79,81]. Active substance (osteoinductive) is composed of bone morphogenetic proteins[36] or growth factors and it is transferred with a carrier that should have osteoconductive properties, be biodisintegrated in a controlled way and be gradually replaced by newly formed bone. Also it must possess mechanical tolerability, it must be capable of releasing its active substances and be easily applied. Carrier's role is to allow bone matrix cells to come in contact with target cells and avert their uncontrolled diffusion in surrounding tissues[32,50,63].
Urist in 1965 and Reddi in 1972 showed that during bone matrix subcutaneous implantation in rats, cellular procedures are presented in a similar way compared to those that occur during intrarticular osteogenesis of fetal tissue and fracture porosis in adults. That is to say, non differentiated mesenchymal host cells are stimulated and differentiated in chondrocytes. Chondrocytes undergo hypertrophy and calcification forming osteocytes that produce new bone. These proteins are osteogenesis proteins or bone morphogenetic proteins (BMPs). They compose a polypeptide family of bone matrix and belong to the hyperfamily of Transforming Growth Factors (TGF-â). The most active of them, BMP-2 is now produced by methods of genetic recombination and in several experimental studies has shown excellent results[72]. Clinical trials of BMP-2 in human are en route.
Furthermore, now days we use several growth factors to produce bone grafts in order to cover osseous defects with very encouraging results. But it is very early to extract conclusions considering their safe and widespread use[10,14,23,37,56,59,62,64].

Some current carrier types are the following:
- Polymers (polylactic-polyglycolic sponges)
- Glycerol (H2O soluble), Gelatin
- Titanium sponges
- Xenografts
- Hydroxylapatites
- Hyaluronic Acid
- Calcium phosphates
- Calcium sulfate

HUMAN GRAFTS
- Osteoconductive
- Osteoinductive
- Osteogenetic

ALLOGRAFTS
- They hold their mechanical tolerances
- They are used in combination with autograft
- They are available in frozen and dry form
Frozen
- They are kept in <60°C
- They have decreased immunological reaction
- They show decreased absorption
- They keep their mechanical tolerances
Dry
- Significantly decreased immunological reaction
- Decreased biochemical tolerances

DEMINERALIZED BONE MATRIX (DBM)
- Grinded cortical bone without its mineral phase
- Demineralization reveals multiple morphogenetic proteins (BMP) and growth factors (GF) and it
- produces a stem cell differentiation
- results in bone production
- exists in proper proportions
- can be wherever used
- Processing combined with carrier differ in
- osteoinduction potential
- osteoinduction confirmation
- osteoconduction potential
- form Ð paste, gel, e.t.c.
- manipulation potentials
- mix with spongy grinds

SYNTHETIC GRAFTS
- Osteoconductive
- Filling of closed osseous defects

POTTERY SUBSTITUTES
- Naturally produced mineral salts processed in >1000°C
- They are relatively tolerant to deformation and absorption

CORAL HYDROXYAPATITE
- They come form Pacific corals
- They are cut in several shapes (limited formation potential)
- They convert triphosphate calcium in slowly absorbed hydroxyapatite
- Osteoconductive
- Increased absorption time

TRIPHOSPHATE CALCIUM
- Paste in injectable form
- Solidification in body temperature
- Mechanical tolerance in compression
- Crystal structure similar to spongy bone

BIOACTIVE POTTERY
-Tablets made from silicon, sodium oxide, calcium oxide and pyrophosphate
- Tablets are combined together and adhere to collagen fibers and growth factors into the infusion area to create a scaffold
- Osteogenesis follows a series of reactions
- Supportive tolerances without structural support

CALCIUM SULPHATE
- With or without an antibiotic
- The only hydrolyzed material in 8-12 weeks
- Bone gaps filling
- Permits vascularization
- Circumferential bone production
- Without any mechanical tolerances
- Carrier for controlled local antibiotic administration

ANIMAL ORIGIN
BOVINE GRAFT
- Without organic phase
- Osteoconductive
- Elimination of the potential for disease transmission due to high temperature processing
Despite the fact that there are no comparing studies from recognized centres and large published series in approved orthopaedic journals referring to documented better outcomes after bone grafts use and to potential complications of their use, however, in USA according to American Academy of Orthopaedic Surgeons in 2002, the bone graft usage per form was the following:
Human Allografts: ~45%
Demineralized Bone Matrix (DBM): ~30%
Synthetic Grafts: ~25%
After a detailed study and data analysis provided by the up to date published studies in approved orthopaedic journals (Seth Greenwald et al, J.B.J.S. Vol. 83A, Suppl. 2-2, 2001), as well as the report of the Committee on biological implants (A.Seth Greenwald, D.Phil. (Oxon), Scott D.Boden, M.D., Victor M. Goldberg, M.D., Yusuf Khan, M.S., Cato T. Laurencin, M.D., Ph.D., Randy N. Rosier, M.D.) in
69th Annual Meeting of American Academy of Orthopaedic Surgeons, Dallas, Texas 2002, the Committee resulted in the following indications for grafts use (Table 2).

CONCLUSION
It is a fact that bone grafts are necessary in several cases in daily orthopaedic practice. However, its application should obey to proper medical indications, avoid abuse and predominantly follow the principle that fracture porosis is independent of bone graft placement, but depends on a variety of factors and mainly on soft tissue and bone biology respect, as well as on principles of stable osteosynthesis. Grafts are secondary to total procedure and not the main operation success factor, thus their utilization should be reasonable and based upon the above principles.

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