CERASORB® & CERACELL®

Innovation That Delivers Osteobiologic Solutions.

The Diamond Concept in Orthopedics, Trauma & Spine

University Medical Center Hamburg-Eppendorf (UKE) / Department of Osteology and Biomechanics / European Society of Tissue Regeneration in Orthopedics and Traumatology (ESTROT)

Bone Regeneration Summit 2018Hamburg, 23-24 March 2018

A new era in bone healingFocus on synthetic bone regeneration materialsA paradigm shift in the treatment of bone defects in orthopedics, traumatology and spine is becoming apparent – with a move away from autologous or xenogenic bone replacement materials towards safe, purely synthetic and biomimetic bone regenerative materials. According to the "Diamond Concept", purely synthetic, biomimetic materials facilitate the interplay of the biological factors relevant for bone healing.

Within the field of surgical orthopedics, the replacement of damaged bone with synthetic biomimetic materials has become increasingly important in recent years. Today's treatments are no longer just about restoring structure, they are also about restoring the function of the damaged area. At the same time, special attention is paid to avoiding the problems associated with biological materials, such as infection, rejection and pain, or inadequate conversion to fully functional tissue. In recent years, a range of methods has been developed to help resolve these problems. An overview of the status quo and the latest developments was provided by the "Bone Regeneration Summit 2018", a certified international continuing education event under the scientific direction of Prof. Dr. Gerald Zimmermann, Mannheim, and Prof. Dr. Michael Amling, Hamburg.

The "Diamond Concept" of fracture healingIn recent decades, one key insight that has widely gained acceptance is that the build-up of new bone depends not only on the presence of certain growth or other factors, but also on the structure of the surrounding areas and on mechanical stability, reported Zimmermann. The game changer in terms of unhindered bone healing is the coordinated interaction of three different biological processes – osteogenesis, osteoinduction and osteoconduction – on the basis of mechanical stability1. At the same time, these four relevant prerequisites for bone healing, also known as the "Diamond Concept" (Figure 1), provide starting points for therapeutic support of the biological healing process using biomimetic osteoconductive bone regeneration materials and osteoinductive materials such as stem cells, BMA (bone marrow aspirate), PRP (platelet rich plasma) or BMP (bone morphogenetic proteins). The osteogenic pillar of the "Diamond Concept" is formed by three major cell types, the bone-building osteoblasts, the bone-resorbing osteoclasts, and the osteocytes, which orchestrate the activity of the two cell types responsible for bone remodelling. The use of remodeling will renew the human skeleton every 6 years, explained Amling. Unhindered remodeling is crucial for bone healing and the incorporation of implants.

Calcium phosphate-based bone regeneration materialsAccording to the diamond model, purely synthetic, resorbable bone regeneration scaffolds such as β-tricalcium phosphate (β-TCP, CERACELL®,

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CERASORB®) establish mechanical stability and, with their three-dimensional, highly porous scaffold structure act as a matrix for ingrowing cells and growth factors, thus serving as a guide rail for regrowth of bone. In the process of osteointegration, the bioceramics combine with the bony edge of the defect and are resorbed parallel to the formation of new bone2. By contrast, persistent defect fillers (biphasic substances, hydroxyapatite or PMMA) impede physiological bone remodelling at the location of the defect, and therefore also the adaptation of the bone to biomechanical loads.

In a study with patients with a tibial plateau fracture (n=52), the β-TCP ceramic CERASORB®, manufactured by curasan, proved to be extremely effective and safe. In 82% of patients, good to excellent functional results were achieved3. In spinal surgery, the synthetic bone regeneration material has proven effective in the form of a foam composite (CERASORB® Foam & CERACELL® Foam). The results of a prospective, open, monocentric study of 34 patients with indications of instrumented lumbar spinal fusion show that the ceramic-collagen foam in combination with autogenous bone material leads reliably to spondylodesis. Intraoperative application was easy and user-friendly4. Data is now also available from the use of CERASORB® in orthopedic surgery, trauma surgery and hand surgery (n=106), which proves its effectiveness and safety over a period of 10 years5. In all three indications, synthetic bioceramics resulted in full osseous healing and good resorption.

Future potential of β-TCP scaffoldsBioceramic scaffolds can do even more. As Freiburg Prof. Dr. Anke Bernstein reported, β-TCP ceramics can also be loaded with antibiotics – a future prospect in revision surgery and osteomyelitis. The release biokinetics depend on the particular process used to load the scaffolds (vacuum or drop loading). Higher interconnectivity and porosity of the carrier material prove more effective, at least in vitro, in terms of the release of antibiotics. Similarly, good results were demonstrated in a new in vitro study on the biokinetic release behavior of antibiotics by PD Dr. Klaus Edgar Roth, Mainz.

In particular, the long-term release of antibiotics using the CERASORB® Foam scaffold must be underlined here.

Against the background of the "Diamond Concept", the loading of the porous β-TCP scaffold with growth factors, PRP (Platelet Rich Plasma) or BMP´s (Bone Morphogenic Proteins) is a promising approach to the support of bone regeneration, reported Prof. Dr. Peter V. Giannoudis, Leeds, UK. This promotes both the migration of progenitor cells and the diffusion of nutrients into the defect, and on the whole establishes favourable conditions for the proliferation, differentiation and survival of the cells6.

“curasan´s proprietary and scientifically proven CERASORB® and CERACELL® β-TCP product portfolio is the ideal biomimetic platform to be combined with growth factors, PRP´s, bone marrow aspirate, stem cells and even antiobiotics in order to address today’s most discussed topics in Orthopedics, Traumatology and Spine,” adds Florian Früh, Head of Product Management at curasan. “The discussion on fusion rates, bone remodeling pace and quality, infection rates, non-unions and osteomyelitis is becoming more and more predominant in the conversation with surgeons. We at curasan believe that both, the industry and science, has to undergo a paradigm shift from pure implantology to a combined approach of disruptive implant and biological treatment therapies in order to address today´s hot topics,” he continues.

“This year we proudly celebrate our 30-Year anniversary. curasan is present in over 50 countries with almost 80 partners, having sold close to 2 million units of CERASORB® with zero reported product related side effects and additionally having published 220 scientific articles on our technologies.” mentions Michael Schlenk, CEO of curasan. “CERASORB® is listed as the international gold-standard for β-TCP´s at the International Center for Diffraction Data since 2005 and is today´s benchmark in the industry in regards to phase-purity,” adds Gregor Siebert, Head of Marketing and Sales. “We don´t stop here but continue to add innovative high-end products to

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our portfolio in order to underline our position as a global technology leader in the field of bone regeneration”, he continues.

curasan develops, manufactures and markets biomaterials and medical devices in the field of bone and tissue regeneration, wound healing and osteoarthritis therapy. As a pioneer and global technology leader in the growing field of regenerative medicine, curasan is specialized primarily on biomimetic bone grafting materials for dental, oral/maxillofacial, orthopedic and spinal applications, i.e. materials mimicking biological structures.

Numerous patents and a broad record of scientific publications demonstrate the clinical success of

curasan´s highly innovative products. Clinicians worldwide benefit from the broad range of the premium quality and easy to use portfolio offered by the technology leader curasan.

curasan maintains its own high-tech facilities for research, development and manufacturing of biomaterials in Frankfurt/Main, Germany. In addition to its headquarters, the company has a subsidiary, Curasan, Inc., in Wake Forest, N.C., USA.

curasan´s innovative products are cleared by the US Food and Drug Administration (FDA) and many other international authorities and available in almost 50 countries worldwide. curasan AG is a public company listed in the General Standard at the Frankfurt Stock Exchange.

References

1 | Giannoudis PV et al. Injury 2007; 38 (Suppl 4): S3-S6

2 | Damron TA. Nanomedicine 2007; 2: 763-775

3 | Rolvien T et al. Knee 2017; 24: 1138-1145

4 | Daentzer, D. Hübner W-D. OUP 2016; 4: 242-248

5 | Gruber A. OUP 2017; 3: 164-171

6 | Lavik E, Langer R. Appl Microbiol Biotechnol 2004; 65: 1-8

Figure 1: "Diamond Concept" of bone healing [after: Giannoudis PV et al. Injury 2007; 38 (Suppl 4): S3-S6]

Osteoprogenitor cells (Osteogenesis)

Scaffolds / Matrix(Osteoconduction)

Proteins / Growth Factors(Osteoinduction,Osteopromotion)

Mechanical Stability

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Prof. Dr. Gerald ZimmermannTheresienkrankenhaus Mannheim, Germany

The Diamond Concept

The principals of bone regeneration

The "Diamond Concept" (Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: The Diamond Concept. Injury. 2007;38:S3-S6) involves four mandatory components for successful fracture healing. First, several osteogenic cell types, primarily osteoblasts, are functioning towards restoring the bone. Second, growth factors are needed to induce a cascade of cellular processes. These growth factors are secreted by endothelial cells, platelets, macrophages, monocytes, but also by mesenchymal stem cells, and osteogenic cells themselves.

Third, mechanical stability is a decisive factor for bone healing and for the formation of a callus that bridges the fracture site and enables the transfer of loads across the fracture line. Last, primarily synthetic osteoconductive scaffolds, such as CERASORB® and CERACELL® Foam or Granules, are needed in large bone defects to ensure fracture healing. An overview of the Diamond Concept is described in Figure 1.

The often-generalized statement of orthopedic surgeons that “if one performs a stabilization via osteosynthesis, the biology would work on its own” is generally a false assumption. All four components of the Diamond Concept need to be considered and aligned when operating fractures, especially in more complicated situations (i.e. revision surgeries, infections).

In case of open, multi-fragment lower limb fractures, this problem can be well demonstrated. After primary external fixation and antibiotic treatment, the surgeon often faces the issue of a big bone defect that has to be taken care of in a second surgery. In these cases, the impaired biology might even overrun the mechanical stability.

The variability in the biology of fracture healing is also illustrated by other examples. In children for example, fracture healing tends to occur very quickly, which is related to a generally higher bone remodeling state. It has also been observed by many orthopedic surgeons, that although doing the same type of osteosynthesis, one patient may heal and another may not. One of the main reasons for non-union is infection. In the tibia e.g., non-union rates are up to 45%. The healing interval may go up to 4 years and differs highly between patients. In concerns of socioeconomic aspects, delayed union is associated with immense costs - 16 weeks of delayed healing cost the US healthcare system around 80.000US$.

Coming back to the Diamond Concept, each of the four parts must be considered individually in a situation of potential delayed union. Bone

Figure 1: The Diamond Concept according to Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: The Diamond Concept. Injury. 2007;38:S3-S6)

Osteoprogenitor cells (Osteogenesis)

Scaffolds / Matrix(Osteoconduction)

Proteins / Growth Factors(Osteoinduction,Osteopromotion)

Mechanical Stability

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grafting is a surgical procedure that is applied in fractures that are more complex and involve big bone defects that need to be bridged to ensure sufficient fracture healing. Bone grafts must be compatible with all four parts of the Diamond Concept. Bone grafts may be autologous (mostly harvested from the iliac crest), allogeneic (cadaveric bone/bone bank), or, increasingly used nowadays, synthetically derived.

The main requirements for bone grafts are osteo-conduction (new bone growth on the scaffold), osteoinduction (cells differentiating into bone forming osteoblasts) and osteogenesis (bone/callus formation). Synthetic bone grafts such as Betatricalciumphospates (β-TCP´s) can be used in patients with complex distal radius or tibia fractures involving the joint surface. Anatomic reduction of depressed joint fragments is the main goal in fracture treatment; and the preservation of the joint surface is one of the greatest challenges. Synthetic β-TCP scaffolds, such as CERASORB® (pure phase β-TCP) or CERACELL® (β-TCP + Si) may help in achieving this goal.

Next to the scaffolds and mechanics, vascularization plays a major role in fracture healing. In the distal tibia, it is very low and is one of the reasons for the high non-union rate. The application of growth factors (i.e. TGFß, BMP2 and BMP7) can be used to promote frac ture healing; however, they are rather expensive. Moreover, there is a huge potential use of mesenchymal stem cells (MSC´s) to further promote fracture healing.

In summary, one has to keep in mind that delayed union or non-union is often observed in infected areas. Therefore, the surgical concept should involve a radical debridement, treatment of the infection with antibiotics, and subsequent osteosynthesis in combination with the use of biomimetic bone substitutes when indicated. In more complex situations and potential risk factors for non-union (i.e., anatomical sites, comorbidities) one should consider additional fracture healing induction from beginning. As stability is one of the most important points in the Diamond Concept, biomimetic bone scaffolds contribute to a better outcome.

Take home messages

Only the combination of a well performed osteosynthesis and the four key factors of the Diamond Concept (mechanical stability, osteoconduction by biomimetic scaffolds, osteoinduction by growth factors and osteogenesis by stem cells) promote the ideal fracture healing.

Synthetic and biomimetic scaffolds like CERASORB® and CERACELL® help in achieving the goals of anatomic reduction of depressed joint fragments and simultaneously preserve the joint surface.

CERASORB® and CERACELL® Foams, Granules and Block Forms can be prophylactically dotted with antibiotics (Gentamycin and Vancomycin) to reduce the potential risk of a surgical site infection or in order to locally treat an infected area.2

References

1 | Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: The Diamond Concept. Injury. 2007;38:P3-P6.

2 | PD Dr. Klaus-Edgar Roth et. al., JGU Mainz, Center for Orthopedic and Trauma Surgery.

Note: It has to be reassured, that the chosen antibiotic does not contain any sulfuric acids: Release of Antibiotics out of a moldable collagen ß-Tricalcium composite compared to two Calciumphosphate Granules. EDTA´s or any other aggressive preserving agents since those could lead to a premature lysis of the β-TCP. For a full positive list of antibiotics, please contact cerasorb@curasan.com

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Prof. Dr. Michael AmlingIOBM Hamburg-Eppendorf, Germany

The Diamond Concept

The principals of bone remodeling – Cellular basis for unlimited regeneration

When talking about bone regeneration, it is important to keep in mind the basic functions of bone and the skeleton in general. Bone enables us to walk and provides protection for the internal organs. Bone is light, resistant, and even forgiving under optimal conditions. Bone is adaptable until old age.

Another important function of bone is to provide the organism with calcium and phosphate. Bone quality consists of the 3 major components: material, structure and cells. Looking inside a bone, microstructure is one important property to check for – this is demonstrated in cases of osteoporosis vs. normal bone structure.

Bone structure is the result of the cellular power of bone-forming osteoblasts, bone-resorbing osteoclasts and matrix-embedded osteocytes

(Figure 1). This can be demonstrated in the lateral views of radiographs from patients with different disease states such as osteoporosis, renal osteodystrophy, spondylodesis or ankylosing spondylitis, where the individual morphological appearance (i.e. sandwich vertebral bodies) is the result of an altered cellular function.

It is all about the three cellular players. Osteoclasts have the only function to resorb bone. Bone structure can only be changed through the initial removal of bone matrix. The function of osteocytes has not been taken into consideration for a long time. Osteocytes are regulators and control the interaction of osteoblasts and osteoclasts. Bone remodeling serves for lifelong regeneration. Therefore, the skeleton is remodeled completely around every 6 years so that your oldest bone is 6 years old no matter how old you are.

Figure 1: Simplified bone remodeling scheme demonstrates the interplay between the bone cells. Ob = Osteoblasts; Oc = Osteoclasts; Ot = Osteocytes; (+) = stimulation; (–) = inhibition.

Ob Oc

Ot

Oc Precursor

Oc

Sclerostin

RANKL

RANK

+

-

Cytokines Vitamin D

+ Differentiation

Differentiation

Fusion

RANKL M-CSF

+

Apoptosis

Osteocalcin

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Several ideas exist about what controls bone remodeling. Is it mainly osteoclasts? Is it the brain? The latter idea originates from Paul Sudeck, who has first described “Sudeck's atrophy” where the central nervous system plays an important role in a regional bone loss. There is strong evidence that the hypothalamus in involved in the control of bone mass1. However, this control mechanism may not be enough – There is a so-called “crosstalk” between osteoblasts, osteoclasts and osteocytes, which is mainly controlled by osteocytes via the secretion of numerous signaling molecules. The osteocyte lacuno-canalicular network – a network of micron-sized pores (lacunae) and nanometer-sized channels (canaliculi) – is the main regulator of bone remodeling and represents the bone’s mechanosensory function2.

The basic aspects of bone regeneration must be translated into clinical practice in order to achieve an optimal outcome i.e., in the use of bone substitutes. From several histological and micro-morphological studies from retrieved specimen we have learned several things regarding the incorporation of bone substitutes such as allografts or synthetic bone grafts. First, bone remodeling with the subsequent interconnection of the host bone and the allograft bone is present in the majority of the interface, leading to progressive incorporation of the allograft. Furthermore, biomimetic bone regeneration materials such as CERASORB® β-TCP are an ideal alternative to autografts and allografts in the restoration of bony defects if the cellular processes mentioned above fully intact3.

Take home messages

Bone structure is the result of the cellular power of bone-forming osteoblasts, bone-resorbing osteoclasts and matrix-embedded osteocytes . It is all about those three cellular players.

The osteocyte lacuno-canalicular network is the main regulator of bone remodeling and represents the bone’s mechanosensory function.

Biomimetic bone regeneration materials such as CERASORB® and CERACELL® are an ideal alternative to autografts and allografts in the restoration of bony defects if the cellular processes mentioned above fully intact.

References

1 | Ducy P, Amling M, Takeda S, Priemel M, Schilling AF, Beil FT, et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell. 2000;100(2):197-207.

2 | Milovanovic P, Zimmermann EA, Hahn M, Djonic D, Puschel K, Djuric M, et al. Osteocytic canalicular networks: morphological implications for altered mechanosensitivity. ACS Nano. 2013;7(9):7542-51.

3 | Rolvien T, Barvencik F, Klatte TO, Busse B, Hahn M, Rueger JM, et al. β-TCP bone substitutes in tibial plateau depression fractures. Knee. 2017.

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Prof. Dr. Björn BusseIOBM Hamburg-Eppendorf, Germany

The Diamond Concept

A biomechanical perspective on bone quality

As a basis for the discussion on the topic of bone quality, this term must be defined. Bone quality is assumed to be a total sum of numerous characteristics. “Quality is the totality of features and characteristics of a product1”. Bala and Seeman talk about bone “qualities” rather than “quality”2. When talking about bone quality, it is important to acknowledge the hierarchical structures of bone – from osteons to collagen molecules. All features contribute to bone quality to some extent. Since 80% of the skeleton’s mass is composed of cortical bone (as opposed to 20% of trabecular bone), it seems logic that cortical bone quality plays a tremendous role in the fracture risk. The assessment of bone quality requires different methods with different resolutions. Measures of bone quality correlate with the risk of fractures.

There are certain age-related changes of cortical bone – the Haversian area increases and the mean wall thickness decreases, while the porosity increases. The origin of bone fracture is dependent on many things. On the one hand, bone loss contributes to increased fracture risk. However, if individual trabeculae from healthy and osteoporosis individuals with the same trabecular thickness are separated, osteoporosis cases fracture, while healthy cases do not. This was found to be due to the increased mineralization

heterogeneity in osteoporosis cases3. Going beyond the bone’s mineral, for example using small angle x-ray scattering, the bone quality up to nanometer scale crystals can be assessed.

Osteocytes, the matrix-embedded well-connected cells orchestrating bone remodeling, are another important determinant of bone quality. An age-related reduction of osteocyte lacunae has been observed previously4. Furthermore, the osteocyte canalicular communication across osteons is disrupted in aged bone. This means hampered nutrition and oxygen supply for the cells. The older an osteon gets, the more the cement lines of an osteon mineralize and this might be another contribution to fracture5. The crack growth of fractures has been associated with bone mineralization, i.e. in vitamin D deficient compared to healthy specimens6 (Figure 1). Next to the mineral components of bone, collagen quality is very important to look at. This comprises e.g. collagen orientation or collagen crosslinking.

In conclusion, bone quality consists of structural properties (geometry, size, microarchitecture) and material properties (mineral, collagen, microdamage). Assessing different qualitative measures of bone quality is important to understand the origin of fracture as well as the extent of bone regeneration.

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Take home messages

Both, structural properties (geometry, size, microarchitecture) and material properties (mineral, collagen, microdamage) together determine the overall bone quality.

The nutrition and oxygen supply for the cells are crucial to the overall bone regeneration process.

Bone is layered in a hierarchical structure, from osteons to collagen molecules. The ratio between cortical and trabecular bone is 1:4, and therefore, the role of cortical bone in determining the bone quality is significant.

Figure 1: 3D reconstruction of the crack path via SRµCT. In vitamin D–deficient samples, the crack takes a winding breaking path across the osteons between the highly mineralized bone and cement lines. (from 6)

References

1 | Bouxsein ML. Bone quality: where do we go from here? Osteoporos Int. 2003;14(5):118-27.

2 | Bala Y, Seeman E. Bone’s material constituents and their contribution to bone strength in health, disease, and treatment. Calcif Tissue Int. 2015;97(3):308-26.

3 | Busse B, Hahn M, Soltau M, Zustin J, Puschel K, Duda GN, et al. Increased calcium content and inhomogeneity of mineralization render bone toughness in osteoporosis: mineralization, morphology and biomechanics of human single trabeculae. Bone. 2009;45(6):1034-43.

4 | Busse B, Djonic D, Milovanovic P, Hahn M, Puschel K, Ritchie RO, et al. Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone. Aging Cell. 2010;9(6):1065-75.

5 | Milovanovic P, vom Scheidt A, Mletzko K, Sarau G, Püschel K, Djuric M, et al. Bone tissue aging affects mineralization of cement lines. Bone. 2018;110:187-93.

6 | Busse B, Bale HA, Zimmermann EA, Panganiban B, Barth HD, Carriero A, et al. Vitamin D deficiency induces early signs of aging in human bone, increasing the risk of fracture. Sci Transl Med. 2013;5(193):193ra88.

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Prof. Dr. Anke BernsteinUniversitätsklinikum Freiburg, GermanyPD Dr. Klaus Edgar Roth Universitätsklinikum Mainz, Germany

The Diamond Concept

Release kinetics of drugs from bioceramic scaffolds & Biokinetic release of antibiotics from biomimetic, pure-phase β-TCP’s – an in-vitro analysis

Release kinetics of drugs from bioceramic scaffoldsMusculoskeletal infections can occur spontan-eously (e.g. arthritis or spondylodiscitis) or as a postoperative complication (e.g. implant-associated infection). Implant-associated infec-tions are mainly caused by Staphylococcus aureus, which can generate biofilms. These biofilms are very difficult to treat and infected bone tissue must often be removed completely by surgical debridement, followed by an antimicrobial treatment over several weeks.

There are certain problems when antimicrobial treatment is necessary. First, the physiological pH-value of 7.4 in healthy tissue decreases to a pH-value of around pH 5.0 in inflamed tissue, which can change the effect of the applied drug. Second, the dosage of parenterally given antibiotics would have to reach toxic serum levels in order attain the necessary therapeutic level on-site. Therefore, locally applied antibiotics have the advantage of reaching high levels without causing systemic side effects (1000-times higher local

tissue concentrations). A scaffold is necessary not only to fill up bone defects but also as a basis for the release of the antibiotics.

In other words, the interest is focused on bone substitute materials, which can release antibiotics. Non-degradable materials require a two-stage operation involving their removal at a second step. Biodegradable and biomimetic bone substitutes do not have to be removed surgically. Calcium phosphate ceramics are well established as an implant material. Their degradability is determined by water solubility and porosity. The lower the calcium/phosphate ratio, the more rapidly the ceramic degrades. Several methods have been described for loading porous ceramics with additives like antibiotics or other drugs.

Regarding the methods for loading calcium phosphate ceramics, short and long-term drug release can be differentiated. By using adhesive loading, no long-term release is possible. In order to assess the possibilities of lengthening the drug release, four types of ceramic granules (curasan

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AG) were studied with two different loading methods1: 1. CERACELL®, a pure-phase β-tricalcium phos-phate (β-TCP) with silicate doting (Si).2. Osseolive®, a calcium-potassium-sodium-phosphate glass ceramic with silicate doting (GC).3. Osbone®, a pure ceramic of hydroxyapatite (HA). 4. CERASORB® Foam (sponge), a highly porous composite of porcine collagen (collagen-complex) and pure-phase β-TCP granules of variable of size and density.

Initially the morphology of the granule carrier systems was examined with ESEM, stereomicroscopy, µCT-imaging and Camsizer® regarding porosity, interconnecting pores and granule size. Method I (ceramic granules, TCP-dowel) was an adhesion based method with drop and dip coating. Method II (TCP- dowel) used a loading chamber with a combination of a hydrogel (alginate) and the different drugs2. Drug release patterns following the drip loading and the vacuum method with vancomycin (VAN) at concentrations of 5 and 50 mg/ml were compared. The influence

of pH 7.4 compared to pH 5.0 on release behavior was studied.

All samples except for the collagen-complex showed an initial VAN burst release with a following steady release (Figure 1). Increasing the porosity and interconnecting structure of, in particular, Osseolive® (GC) and CERACELL® (β-TCP + Si) could improve release concentration, duration and stability.

On day 14 of the samples series with VAN 5 mg/ml, a release could be measured only from the sponge. When loaded with 50 mg/ml VAN, the samples all evidenced a high release until the last day of 15 sampling, the highest concentration being released from Osbone® (HA). The method of drip loading was superior to vacuum loading. Only the superficial area of the ceramic granules could be loaded.

With drip loading, the granules are given a longer time of incubation in the antibiotic solution and can consequently absorb more of the drug.

Figure 1: Cumulative release after drip leading and vacuum loading with VAN 5 mg/ml and 50 mg/ml from a) β-TCP, b) Osseolive®, c) Osbone®. From (1)

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Biokinetic release of antibiotics from biomimetic, pure-phase β-TCP’s – an in-vitro analysis The major goal in the treatment of osteomyelitis is a good bone substitution and a high antimicrobiotic level, which can only be achieved by locally applied antibiotics due to the diminished local vascularity in osteomyelitis. Different methods and various grafting materials are used for the purpose of bony reconstruction. Synthetic calcium phosphate bone graft materials, which have excellent biocompatibility, are commonly used as alternatives to autogenous bone.

To test for the release of antibiotics from β-TCP, 5 pellets of CERASORB® and CERASORB® M were soaked for 1 minute in a gentamicin or vancomycin solution (40 mg/ml)3. The antibiotic elution and concentration of gentamycin and vancomycin were measured using photometrically-based measurement and homogeneous particle-enhanced turbidimetric inhibition immunoassays (PETINIA). Initially both materials showed a high release of the loaded antibiotics, CERASORB® M showing a lower initial release level for gentamicin and vancomycin than CERASORB®. At day 4 the

gentamicin concentration of the CERASORB® M granules was under the detection threshold, for CERASORB® the gentamicin concentration was undetectable at day 6. The vancomycin release-level followed a similar pattern, although the vancomycin concentration eluted by CERASORB® M granules stayed above the detection threshold during the period of the experiment.

Granule/collagen-composites (CERASORB® Foam) offer a higher loading capacity for antibiotics and a stepwise degradation kinetic which may lead to a stronger and longer lasting active agent release1. For CERASORB® granules and hydroxyapatite granules, there is a change point of the antibiotic release, with a steep decline of release within the first 3 days. CERASORB® Foam has a more steady release for 9 days. The single use of collagen matrices seems to show similar characteristics in release kinetics as it is observed for granules and beads. The combination with TCP provides the long-term release. In summary, it can be stated that β-TCP-collagen composites compared with β-TCP- and hydroxyapatite granules offer a significantly stronger and longer release of gentamycin and vancomycin.

References

1 | Faigle G, Bernstein A, Suedkamp N, Mayr H, Peters F, Huebner W, et al. Release behavior of VAN from four types of CaP-ceramic granules using various loading methods at two different degrees of acidity. J Mater Sci Mater Med. 2018;29(1):12.

2 | Seidenstuecker M, Ruehe J, Suedkamp NP, Serr A, Wittmer A, Bohner M, et al. Composite material consisting of microporous β-TCP ceramic and alginate for delayed release of antibiotics. Acta biomaterialia. 2017;51:433-46.

3 | Maier GS, Roth KE, Andereya S, Birnbaum K, Niedhart C, Lühmann M, et al. In vitro elution characteristics of gentamicin and vancomycin from synthetic bone graft substitutes. The open orthopaedics journal. 2013;7:624.

Note: Osseolive® is not yet commercially available.

Take home messages

Antibiotic drip loaded biomimetic bone scaffolds are not only necessary to fill up bone defects but also as a basis for the release of the antibiotics in osteomyelitis and spondylodiscitis.

An increased porosity and interconnecting structure of a biomimetic bone graft substitute such as CERASORB®, CERACELL® improve antibiotic release concentration, duration and stability.

A stronger and longer release of antibiotics (Gentamycin and Vancomycin) has been seen in β-TCP and collagen composites as compared to β-TCP and HA composites

Figure 1: 42 year old male 6 months post fixation (left) and osseus union after BMP administration.

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Prof. Dr. P. Giannoudis University of Leeds, United Kingdom

The Diamond Concept

The role of combining growth factors with bioceramic scaffolds for bone regeneration

When combining growth factors and bioceramic scaffolds it is first important to talk about both of these factors alone. Growth factors include demineralized bone matrix (DBM), platelet rich plasma (PRP), and bone morphogenetic proteins (BMP´s). Bone formation by autoinduction is a well-known mechanism that was first described in Science in 19651. Target mesenchymal stem cells (MSCs) perform mitogenesis and differentiation with the presence of growth factors.

Do growth factors work when administered without a scaffold? In some cases they do. For example in an open tibial fracture, BMP 7 led to osseous union (Figure 1). In a femoral non-union, the osteosynthesis was revised, cleaned and BMP was administrated. This also led to union. Therefore, it can be stated that BMP´s promote a successful bone repair at least in some cases.

When comparing growth factors and autologous bone grafts, it has to be considered that growth factors are produced by DNA recombinant technology. They have only one property being osseoinductive. However, they mostly need a carrier, which can be autologous bone or a biomimetic scaffold. Autologous bone has the major advantage that it is naturally available, no carrier is required and that it possesses all three important properties osteogenicity, ostoinductivity and osteoconductivity. When using BMP alone, bone defects of up to 2 cm can be successfully treated. Beyond that, some type of scaffold is needed. The limitations of BMP´s further include the high costs, containment, carriers, ideal dose, and timing of implantation.

A biomimetic scaffold, such as CERASORB® or CERACELL®, is a structure that possesses the necessary properties that support the attachment of bone forming cells for subsequent bone formation. The overall shape of the biomimetic scaffold represents the superstructure, the cellular level structure of the surface represents the microstructure, and the subcellular level structure of the surface represents the nanostructure2. There are various scaffold types such as ceramics, polymers (biological, synthetic, and collagen hybrids), poly-lactic-co-glycolic acid family (PLA, PGA, PLGA), polyurethane family and hydrogels. Bioceramics are further divided in three groups bioinert, surface, bioactive and bioresorbable such as (β) tricalcium phosphate.

6 months post fixation 4 months after BMP use 6 months post fixation 4 months after BMP use 6 months post fixation 4 months after BMP use

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The advantages of ceramic based biomaterials are e.g. the reproducible quality in terms of phase-purity and porosity, the versatility of sizes and shapes, the fact that Ca-P compounds are abundant in nature with different Ca/P ratios, chemical composition and/or crystal structures and that there is strong compositional resemblance to bone mineral (around 60~70 wt% of human bone mass is made of a Ca-P mineral with a chemical composition similar to HA).

Combining growth factors with biomimetic, bioceramic scaffolds, such as CERASORB® or CERACELL®, ultimately make sense. According to the Diamond Concept3 both, biomimetic scaffolds and growth factors are one of the minimum requirements that have to be present

for bone repair to be initiated and successfully terminated. In this context, bioceramics:a. facilitate cell attachment and migrationb. retain cells and molecular mediatorsc. enable diffusion of nutrients and productsd. exert certain mechanical and biological influences influencing cellular elements ande. incorporate desirable biological and chemical signaling.

In other words: Scaffolds should provide a “friendly environment for growth factors”. Indeed, the successful use of biomimetic bioceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for BMP has been studied and confirmed4.

References

1 | Urist MR. Bone: formation by autoinduction. Science. 1965;150(3698):893-9.

2 | Norman JJ, Desai TA. Methods for fabrication of nanoscale topography for tissue engineering scaffolds. Ann Biomed Eng. 2006;34(1):89-101.

3 | Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the Diamond Concept. Injury. 2007;38:S3-S6.

4 | Alam MI, Asahina I, Ohmamiuda K, Takahashi K, Yokota S, Enomoto S. Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2. Biomaterials. 2001;22(12):1643-51.

Take home messages

Although, BMP´s when used as stand-alone can treat bone defects of up to 2cm, they mostly need a carrier which can be autologous bone or a biomimetic scaffold such as CERASORB® or CERACELL®.

The reproducible quality in terms of phase-purity and porosity as well as the versatility of sizes and shapes are clear advantages of ceramic based biomaterials such as CERASORB® or CERACELL®.

Biomimetic scaffolds (β-TCP and/or composites) in combination with growth factors are one of the minimum requirements that have to be present for bone repair to be initiated and successfully terminated. This has been studied and confirmed.

CERASORB® & CERACELL® – Always First Choice.

curasan AGLindigstrasse 4 63801 KleinostheimGermanycerasorb@curasan.comceracell@curasan.comwww.curasan.com

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CERASORB® & CERACELL® bone regeneration materials. Innovation That Delivers Osteobiologic Solutions.

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