Bone augmentation by replica-based bone formation.

OBJECTIVE: The sources of iliac crest bone grafts are limited. Alternatives are evaluated due to the progress in biomaterial sciences. Synthetical hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP) or biphasic compounds, or even a mélange of HA and ß-TCP will replace bovine ceramics. The goal is maintenance of replica-based-bone formation (RBBF) for bone augmentation. METHODS: 2 female and 2 male patients between 41 and 73 years with 5 sinus elevations were evaluated. Sinus elevations with lateral fenestration, trapezoidal-muco-periosteal flaps and filling with micro-chambered beads (1.5 mm) was performed. A porcine-collagenous membrane and the refixated flap covered the defect. A biopsy program over 20 months was confirm confirm the maintenance of the newly formed bone. RESULTS: A fast bone formation was pronounced. The biopsies revealed mature lamellar bone and full osseointegration of the ß-TCP implant. The biopsy after 20 months showed compact bone with osseointegration of minor rests of the ceramic implant. The defect revealed a mature bone stock already after 5 weeks. SIGNIFICANCE: The introduction of the replica-based-bone formation (RBBF) around micro-chambered beads will change the paradigm of bone augmentation. The next step of the ongoing study has to redefine the interval for implant insertion. The clinical approach confirms the breakthrough to primary mature lamellar bone formation and will permit reduction of placement time for a dental implant.

Dose-dependent osteoinductive effects of bFGF in rabbits.

Growth factors lead to the induction of tissue regeneration in bone healing when coated on biomaterials. Basic fibroblast growth factor (bFGF) combines osteoinduction and neoangiogenesis. This study evaluated bFGF-coated hydroxylapatite implants in two experimental groups with 10 or 100 microg (n = 5 per group) compared with uncoated control implants in the rabbit patellar groove model. We observed an unexpected ineffectiveness compared to the control groups with no significant difference of bone growth after 35 days. However, all samples from the 100 microg experiment (control and coated implant) showed significantly stronger 19-25 day label than both 10 microg groups (control and coated implant). Earlier bone labels are stronger in the 10 microg group with equal observation of similarity between experiment and control site and may indicate a possible inhibitory effect of the higher dosing or osteoclast induction. This result indicates a possible systemic effect of the transient growth factor coating.

Methotrexate-loaded polymethylmethacrylate bone cement for local bone metastasis therapy: pilot animal study in the rabbit patellar groove.

Local chemotherapy is an option in bone metastasis treatment. On the other hand, evaluation of side effects on bone is a difficult issue in drug development and biomaterials research. Most animal models to date are inappropriate or not feasible. Rabbits are an alternative to other bigger mammals and have an appropriate bone structure compared to rats and mice. The patellar groove model of the rabbit offers a standardized and sensitive model to evaluate bone-compromising drug effects. We tested the system with a reproducible polymethacrylate implant as carrier and loaded this system with methotrexate. Three methotrexate concentrations were used in the test samples (250 mg, 1 g and 4 g per 40 g polymethacrylate bone cement; 2 per group) for a descriptive histology approach. Eight German giant rabbits were operated on both knees and received test and control implants in a randomized manner in this bilateral study design. The histological results after 35 days were convincing, encouraging the use in preclinical drug development and biomaterials research and warranting further development of this metastasis treatment approach.

Histological examination of drill sites in bovine rib bone after grinding in vitro with eight different devices.

The way in which bone is processed may affect the quality of the specimen and how much information may be gleaned on histological examination. We investigated eight widely used rasps and drills and compared the results. All large chip cutters damaged the bed and marrow of the bone. The tool that caused the least damage was the wet grinding diamond tool.

Biological effects of shock waves: in vivo effect of high energy pulses on rabbit bone.

Extracorporeal shock waves have recently been introduced to treat pseudarthrosis and aseptic bone necrosis. Only little information exists up to now about the morphological effects of shock waves on normal bone. To study both their acute effect on bone and their long-term effect on its remodelling, 1500 shock waves generated with a Dornier XL1 experimental electrohydraulic lithotripter were applied at 27.5 kV to 19 rabbits divided into five groups. Changes were evaluated after 6, 11, 41, 59 and 85 days. The discharges were focused to the right femur 1 cm above the knee joint. Bone remodelling was assessed in four groups by four-colour fluorescent labelling with labels administered sequentially over 8-day periods during the first month after shock-wave application. Radiographs were taken at dissection to detect fractures. As a result, shock waves were found to induce periosteal detachment with subperiosteal haemorrhages and to press marrow contents out of the medullary cavity. In the medullary cavity, diffuse haemorrhages, haematomas and foci of fractured and displaced bony trabeculae were found. The bone cortex and the knee joint were normal. Radiographs showed lucencies in the marrow but no fractures. During the weeks following shock-wave application, there was intense apposition of new cortical bone resulting in considerable cortical thickening while trabecular remodelling in the medullary cavity was only minor. The displacement of bony trabeculae and marrow contents point to the action of cavitation as the major mechanism of shockwave damage to bone.

[Histomorphology of the bone-cement contact. Experimental phenomenology of the osseous reconstruction processes. I]. / Histomorphologie des Knochen-Zement-Kontaktes. Eine tierexperimentelle Phänomenologie der knöchernen Umbauvorgänge.

In animal experiments the femurs of 25 rabbits were filled with bone cement. In cross sections of the diaphysis even after only one week, small gaps were found filled by lamellar formation of new bone. Osteoblast layers directly on the cement surface were also observed. After 4 to 6 weeks there was a circularly closed contact of inner lamellae and after 9 months a secondary medullary cavity was formed. At this time there was formed a trabcular contact with the implant, interrupted by medullary spaces full of cells. Sometimes woven-bone formations were found in the bone-cement interface.

Modern cementing techniques. An experimental study of vacuum insertion of bone cement.

The results of these experiments show that an increase in the intramedullary pressure (IMP) can lead to embolization of bone marrow contents via the venous drainage system along the linea aspera. A vacuum applied distally to the medullary canal is very effective for filling the diaphyseal tube with cement. The cancellous bone honeycombs of the proximal metaphysis, however, can only be filled if the bone sponge is tunneled at the level of the femoral calcar; a proximal vacuum then yields filling of the cancellous bone framework with bone cement. In order to fill the weight-bearing spongious framework of the pelvic bone with cement, the acetabular cavity should be sealed with a rubber ring and vacuum applied proximo-laterally to the ilium, thereby giving an extremely high suction pressure.

[Primary cancellous bone formation around micro-chambered beads]. / Formación de hueso esponjoso con esferas microcompartimentales.

OBJECTIVES: The question has been raised whether benign bone defects in patients can be treated with bone forming osteoconductive ceramics achieving primarily a cancellous bone scaffold, which is under load from the beginning. MATERIAL AND METHODS: Ten reconstructions were performed in 9patients (6women and 3male), with a mean age of 49 (25-65)years, suffering a high variety of epi- and metaphyseal defects, four tibial fractures, two calcaneal fractures, one pathological phalangeal fracture, one chondroma of the distal femur and two open-wedge osteotomies were filled with micro-chambered ceramic beads of 4 and 6mm in diameter. The mean follow up was 22 (7- 8)months. X-rays and CT-scans formed the basis for the evaluation of the reconstruction of the cancellous bone scaffolds. RESULTS: All cancellous structures were rebuilt, if completely filled with bone-forming elements. If the filling was incomplete, no physiological cancellous bone scaffold resulted. The ß-TCP micro-chambered beads were completely reabsorbed or sandwich-like incorporated at the time of evaluation. The HA micro-chambered beads revealed a contrast enhancement and were integrated in the osseous construction of the bone scaffold. CONCLUSION: Primary cancellous bone formation can be achieved with osteoconductive ceramic micro-chambered beads and can be combined with any osteosynthesis for stable fixation.

Primary cancellous bone formation with BMP and micro-chambered beads: experimental study on sheep.

PROBLEM: The physiological reconstruction of cancellous bone defects in surgery of the locomotor system is an unsatisfactorily solved problem. AIMS: The aims of this study are to examine whether micro-chambered ß-tricalcium-phosphate (ß-TCP) beads provide a certain capillary force suctioning in blood and bone marrow thus forming a stable "negative"-replica of the bone marrow spaces. If so, a new approach for osteoconduction would yield primarily a scaffold of lamellar cancellous bone under load without a long-lasting remodeling process. Recombinant human bone morphogenetic protein (rhBMP) might even enhance all processes of defect healing, remodeling and ß-TCP resorption; gentamicin-loaded ε-caprolactone might protect the implant. MATERIAL AND METHODS: Ten sheep were operated on; the patella-groove model and the tibial head were used. A defect of 9.4 × 20 mm was created using wet-grinding-diamond instruments. Micro-chambered ß-TCP-beads of 4-6 mm with 0.35 mg rhBMP-7 + 0.1 g collagen per animal, or 1.5 g demineralized bone matrix (DBM) paste on the contra-lateral side were implanted. Both osteoinduction groups were compared with the defect in the tibial heads where plain micro-chambered ceramic beads were inserted. Added to the beads was 12.5 mg gentamicin sulphate in 12.5 mg ε-caprolactone-carrier. Outward diffusion was prevented using a 1-mm-thick press-fit inserted ceramic lid. The bone healing, remodeling and resorption of the ceramic in a right-left comparison of the patella groove and the tibial head was examined at 6 weeks, 2 and 3 months; one animal in reserve was followed for 14 months. The animals were perfusion-fixed, the vasculature micro-casted with an acrylate and nondemineralized processed, and with µ-CT and microscopically documented. RESULTS: A primary load-bearing spongiosa had developed around the beads, which shortened the remodeling process. The strong micro-chambered, resorbable ß-TCP-beads demonstrate high capillary strength, resorb blood and bone marrow, and represent a stable formative material which, as a carrier for the controlled local release of BMP, that accelerates bone healing, shortens resorption and remodeling compared with plain and DBM loaded implants. CONCLUSION: Micro-chambered beads represent the bone-forming element, BMP yields a fast defect healing and enhanced remodeling of bone and resorption of ß-TCP compared to delayed and incomplete reconstruction and resorption of ß-TCP on the DBM-side, the plain implants reached nearly the same reconstruction, but far later compared with the BMP loaded implants.

How bone forms in large cancellous defects: critical analysis based on experimental work and literature.

The behaviour of physiological biomaterials, ß-tricalciumphosphate and hydroxyapatite, is analysed based on current literature and our own experimental work. The properties of graft substitutes based on ceramic materials are clearly defined according to their scientific efficiency. The strength of the materials and their biodegradability are still not fully evaluated. Strength and degradability have a direct proportional relationship and are considered the most efficient way to be adapted by their properties to the needs for the treatment of bone defects. New technologies for the manufacturing process are presented that increase those properties and thus open up new indications and easier application of the ceramic materials. The implantation process as well is carefully validated by animal experiments to avoid failures. Based on the experiments, a completely new approach is defined as to how primary bone formation with osteoconductive ceramics can be achieved. The milestones in that approach comprise a synthetically manufactured replica of the bone marrow spaces as osteoconductive ladder, whereas the bead is defined as bone-forming element. As a result, materials are available with high strength if the ceramic is solid or highly porous and possesses a micro-structure. The injection moulding process allows for the combination of high strength of the material with high porosity. Based on the strong capillary forces, micro-chambered beads fulfil most expectations for primary bone formation in cancellous bone defects, including drug delivery, mechanical strengthening if necessary, and stable implantation in situ by coagulation of the blood and bone marrow suctioned in.

Studying bone regeneration with the scanning electron microscope.

The interest in bone structure is very old. That is shown in a historical review with special regard to bone fractures and gap healing. The three-dimensional architecture of the supporting tissue corresponds to the strain acting on it. This can only be presented clearly in the SEM, especially by stereoscopical documentation. Three aspects of utmost importance when studying bone formation were taken into consideration: the three-dimensional presentation of the trabeculae, the processing of the vascular system together with the bony trabeculae, and the preservation of the cells and fibres in their natural state which is only guaranteed in a non-dehydrated frozen state. Reliable bone histology requires the preparation of non-decalcified specimens which are processed for light microscopy. Labeling newly formed bone with fluorochromes is a very common method to analyse sequences of bone formation. Studying the same specimens using both fluorescence microscopy and the SEM provides information not obtained using one method alone. Fresh specimens can be processed for the SEM following light microscopical documentation especially in incident fluorescent light; embedded blocks used for light microscopical investigations can also give valuable information in the SEM when the resin has been removed. The combination of the different methods is of special interest for orthopaedic basic research.

Gap healing of compact bone.

Standard-sized holes were drilled in the tibia of rats resulting in a reproducible model which can be used to study the different stages of gap healing where practically no influence from biomechanical forces is present. Primary bone healing takes place in holes up to a diameter of 800 micrometer. In this case, no differentiated cartilage or connective tissue is formed. The medullary cavity is closed by a sealing callus. The bony splinters resulting from the operation are incorporated into the osseous framework. Revascularization results from the formation of medullary sinus which grows into the hole. The periosteal osseous depositions are supplied by the nutrient vessels, too. The centrifugal arterial bloodstream and the centripetal venous backflow are maintained under stress conditions during gap healing. Our investigations showed that after ten days drill holes in compact bone had been two-thirds filled from the medullary canal. At this point, the periosteal new bone formation had not yet reached the edge of the hole. The medullary sinus system in the medullary canal is different from the periosteal vascular network in that the former displays vessels with a regularly developed framework.

The vascular system of bone marrow.

Not only the arterial, but also the low pressure system of the bone marrow can be demonstrated by micro-corrosion casts using resins of low viscosity. Vascular bone specimen are obtained by injection of self-curing resin and through subsequent maceration. The three-dimensional representation of the vascular pattern in bone marrow in the scanning electron microscope (SEM) enriches the interpretation of morphology and function of the low pressure system. The nutrient arteries enter the medullary canal and then progress in a spiral form branching into the metaphysis. The arterioles arise from the smaller arteries, further divide into smaller arterial capillaries which then drain into sinusoids which were conically enlarged. The three-dimensional and often hexagonal arrangement of the vascular framework is very evident. Increasing in width the marrow sinusoids drain into wider veins and lastly into the central venous canal. Apart from these medullary sinusoids, finely calibered thin-walled venous capillaries in a regularly anastomosing network can be found as an indication that the wide medullary sinusoids are to be considered as a functional state of active bone marrow.

Ice crystal damage in freeze-dried articular cartilage studied by scanning electron microscopy.

The aim of preparing specimens at very low temperatures is to maintain biological tissue in its natural state. The problems which arise are: 1) obtaining the highest possible cooling rate when freezing the tissue; 2) preventing melting with recrystallization during the drying process; and 3) preventing the dried specimens from water vapor contamination. Biological tissue is prepared in a cooling chain by freezing the samples in a supercooled nitrogen bath, breaking the specimens in the frozen state, sputtering the fracture surfaces in a vacuum on the cooling stage of a special device and then transferring the specimens under vacuum to the SEM where they are studied on a cooling stage. They have smooth surfaces, with the liquid and solid phases present simultaneously, and no artefacts. Any shifts or deformations which may occur within the structure cannot yet be determined. Freeze-drying quenched specimens at various temperatures (-140 degrees to -60 degrees C) taking the dynamics of vapor pressure into consideration shows that sublimation, the melting processes with recrystallization and the resulting damage to the structures occur simultaneously during the drying process. The destruction of the tissue is inversely proportional to the ratio of sublimation to melting.

[The significance of blood vessels on both sides of the growth plate]. / Die Bedeutung der Blutgefässe auf beiden Seiten der Wachstumsfuge.

A new 3-D presentation of the whole vasculature of the epiphyseal growth plate clearly allows for a distinction between arterial and venous sections of the blood circulation. Histological investigations using these processing methods could neither prove the connection between epiphyseal and metaphyseal vessels through the growth plate in newborns nor the concept of an arterial epiphyseal blood supply of the growth plate presented by Trueta. It is rather a question of an arterial metaphyseal perfusion and an epiphyseal venous drainage system.

The role of the vessels in the growth plate: morphological examination.

Combined methods of light microscopy to present ossification zones by means of fluorochrome dyes make it possible to explain the contradicting presentations of the vascular system of the growth plate in the literature. The vascular system was casted with methacrylates which can be presented in the scanning electron microscope 3-dimensionally together with the trabeculae as a result of their resistance in the electron beam. The 3-dimensional presentation in the electron microscope allows a clear distinction between the various vascular sections in the arterial flow system. In the microcorrosion casts the vessels of the epiphyseal side of the growth plate can be clearly distinguished from those of the metaphyseal side. The combination of both methods: labelling with fluorochromes investigated in the incident fluorescent light and casting of the vessels studied in the SEM shows close connection between the arterial vascularization and osteogenesis. These findings also explain the reactions on the part of growth behavior following traumatic injuries to the growth plate - reactions which could not be clearly explained up until now. Our findings do not contradict results of studies in the literature. They permit a uniform interpretation of these findings, however. Presentations of the venous drainage system on the epiphyseal side of the growth zone have not been made to date.

Adherence and growth of bacteria on bone cement in vitro and in chronologically infected artificial joints.

Low grade infections following total hip replacements manifest themselves late after surgery. These infections seem to be sterile; there are major problems in identifying the causative organism by cultivation. SEM investigation of small samples of the bone cement, the task of which is to anchor the prosthesis in the bone, is a suitable method for identifying the bacterial origin of these infections. During conventional histological preparation polymethacrylates are dissolved. Freeze-drying techniques, however, allow the preparation of bone cement samples for the SEM. The growth of staphylococci on cement particles shows that these resins can no longer be considered biologically inert. The surface is attacked by the bacteria.

[Morphology and clinical aspect of the lateral ligaments of the ankle joint (author's transl)]. / Morphologie und Klinik des fibularen Bandapparates am oberen Sprunggelenk.

The direction of movement and the range of movement of a joint are determined by the lengths of the ligaments and the positions of their origins and insertions. It follows that any operation which alters the length of a ligament or moves one of its points of attachment is unphysiological. Reconstruction should be carried out with due regard to the normal anatomy. In the ankle joint, for example, the tip of the lateral malleolus should be left free of ligamentary insertions. Until now, anatomical illustrations have shown the lateral ligaments of the ankle joint to be attached to the anterior border of the fibula and to its anterior and lateral surface. All the older illustrations emphasize a band of fibers which passes directly from the neck of the talus to the calcaeus. This band varies in thickness and always forms the base of the triangle formed by the anterior talofibular ligament and the calcaneofibular ligament. Examination of surgical specimens and amputated ankle joints shows that the lateral tip of the fibula is free of ligamentary insertions. The lateral fibular ligaments are attached to the distal end of the fibula around the border of the cartilaginous foint surface. The only fibers which are derived from the lateral surface are those from the periosteum. This fact is of considerable importance in reconstructive surgery of the ankle joint, since strong ligamentary insertions which surround the lateral surface of the fibula necessarily oppose rotation or screw-like movement of the fibula about its longitudinal axis.

The John Charnley Award. Prophylaxis of fat and bone marrow embolism in cemented total hip arthroplasty.

The efficiency of a new cementing technique developed to prevent the risk of intraoperative pulmonary embolism was assessed. Seventy patients with coxarthrosis entered into a prospective, randomized clinical trial. In the control group of 35 cases the total hip replacement was cemented conventionally. In the second group a proximal drainage placed along the Linea aspera, and a distal drainage placed in the diaphysis, created a vacuum in the medullary cavity of the femur during the insertion of the stem. The operation was performed with the patient under blood gas analysis and hemodynamic and transesophageal echocardiography monitoring. Severe transatrial embolic events were observed during the insertion of the femoral component in 94% of the cases of the control group and in 14% of the cases of the vacuum group; the difference is statistically significant. A significant decrease of arterial partial pressure of O2 (-40.8 mm Hg) and increase of the pulmonary shunt values (+28.3%) occurred 5 minutes after the observation of embolic events in the cases operated on conventionally, but these parameters showed minimal changes in the vacuum group. The rise of intramedullary pressure in the femur is the most decisive pathogenic factor of pulmonary embolism during total hip arthroplasty. The logical prophylactic measure to prevent intravasation of fat and bone marrow is to create sufficient drainage. The cohorted investigation showed the value of the vacuum cementing technique for a substantial reduction of intraoperative embolism and pulmonary impairment.

[Anatomic basis of the cemented femur shaft. A comparative study of straight and anatomic design]. / Die anatomische Basis des zementierten Femurstieles. Eine Vergleichsstudie zum geraden und anatomischen Design.

AIM: The purpose of this study was to compare cemented anatomic stems with cemented straight stems regarding cement mantle and stem orientation in the medullary canal. METHODS: In a cadaver study, 10 anatomic SP II stems and 10 MEM straight stems were implanted in paired human femora using a standardised modern cementing technique. In one group the femoral canal was prepared using conventional broaches, in the other group diamond hollow-cutters were used. Standardised horizontal cuts were made and evaluated regarding stem orientation and cement mantle using digitised image analysis. RESULTS: All SP II stems in the "diamond group" showed good stem centralisation without cement mantle defects. In the "broach group" all diaphyseal cancellous bone had been removed and only 3/5 stems were well orientated. No reproducible stem centralisation was achieved with the straight stems. All stems showed an oblique orientation from antero-proximally to postero-distally with direct stem to bone contact. The variation from the optimal stem alignment along the canal axis showed a mean deviation of 3.1 mm in the anatomic stem group and 10 mm in the straight stem group. The canal preparation using broaches showed frequent fractures of the cancellous bone and debris interposition despite the use of jet-lavage. Most of the cancellous bone in the diaphysis had been destroyed. CONCLUSIONS: Anatomic stems show a better stem centralisation and a more even cement mantle than straight MEM stems. The use of high volume straight stems carries a significant risk of producing cement mantle defects. Diamond instruments are less traumatic than broaches for femoral canal preservation and help to preserve diaphyseal cancellous bone, which improves stem self-centralisation.