The ability to edit DNA at the nucleotide level using clustered regularly interspaced short palindromic repeats (CRISPR) systems is a relatively new investigative tool that is revolutionizing the analysis of many aspects of human health and disease, including orthopaedic disease. CRISPR, adapted for mammalian cell genome editing from a bacterial defence system, has been shown to be a flexible, programmable, scalable, and easy-to-use gene editing tool. Recent improvements increase the functionality of CRISPR through the engineering of specific elements of CRISPR systems, the discovery of new, naturally occurring CRISPR molecules, and modifications that take CRISPR beyond gene editing to the regulation of gene transcription and the manipulation of RNA. Here, the basics of CRISPR genome editing will be reviewed, including a description of how it has transformed some aspects of molecular musculoskeletal research, and will conclude by speculating what the future holds for the use of CRISPR-related treatments and therapies in clinical orthopaedic practice.

Cite this article: Bone Joint Res 2020;9(7):351–359.

Objectives

MicroRNAs (miRNAs) have been reported as key regulators of bone formation, signalling, and repair. Fracture healing is a proliferative physiological process where the body facilitates the repair of a bone fracture. The aim of our study was to explore the effects of microRNA-186 (miR-186) on fracture healing through the bone morphogenetic protein (BMP) signalling pathway by binding to Smad family member 6 (SMAD6) in a mouse model of femoral fracture.

A heavy infiltrate of foreign-body macrophages is commonly seen in the fibrous membrane which surrounds an aseptically loose cemented implant. This is in response to particles of polymethylmethacrylate (PMMA) bone cement and other biomaterials. We have previously shown that monocytes and macrophages responding to particles of bone cement are capable of differentiating into osteoclastic cells which resorb bone. To determine whether the radio-opaque additives barium sulphate (BaSO. 4. ) and zirconium dioxide (ZrO. 2. ) influence this process, particles of PMMA with and without these agents were added to mouse monocytes and cocultured with osteoblast-like cells on bone slices. Osteoclast differentiation, as shown by the presence of the osteoclast-associated enzyme tartrate-resistant acid phosphatase (TRAP) and lacunar bone resorption, was observed in all cocultures. The addition of PMMA alone to these cocultures caused no increase in TRAP expression or bone resorption relative to control cocultures. Adding PMMA particles containing BaSO. 4. or ZrO. 2. , however, caused an increase in TRAP expression and a highly significant increase in bone resorption. Particles containing BaSO. 4. were associated with 50% more bone resorption than those containing ZrO. 2. . Our results suggest that radio-opaque agents in bone cement may contribute to the bone resorption of aseptic loosening by enhancing macrophage-osteoclast differentiation, and that PMMA containing is BaSO. 4. likely to be associated with more osteolysis than that containing ZrO. 2.

1. Cell differentiation around screws manufactured by two American and two Swiss companies and inserted into seventy femora in forty-one adult mongrel dogs has been observed over periods varying between two weeks and nine months. 2. This study reveals that, despite their excellent holding power, such screws are not everywhere in firm contact with the surrounding bone at the time of insertion. Indeed, only part of the thread surface facing the head of the screw touches the compact bone, all other surfaces being separated by a space up to 150 µ in thickness. 3. These spaces result both from the surgical technique employed and from the inaccurate measurements of drills, screws and taps. 4. Migrating cells invade these spaces during the first two weeks. In the absence of movement, these cells differentiate into osteogenic cells; movement leads to differentiation into fibroblasts, chondroblasts and osteoclasts, and failure of fixation ensues. In contrast, callus formation by osteogenic cells firmly anchors screws in four to five weeks, well before callus uniting the bone fragments has been established. 5. Extremities should be protected from undue stresses during those first few weeks after osteosynthesis, whatever the technique. 6. This study clearly demonstrates the importance oftesting screws in living bone to ascertain their holding power at all stages of fracture healing

Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages

1. Histochemical staining and correlated biochemical estimations of five hydrolytic enzymes were done on eighteen benign and twenty malignant fibroblastic lesions of bone and soft tissue. 2. Alkaline phosphatase activity was moderate in a fibroma and very high in fibrous dysplasia. In a typical fibrosarcoma the fibroblasts showed no enzyme activity and estimations were low. Exceptions indicated an osteogenic potential in the tumour. 3. ß-glucuronidase, leucine aminopeptidase, and to a less extent non-specific esterase, were more active in malignant than in benign lesions, and the highest activities were found in sarcomata arising in Paget's disease of bone. 4. Acid phosphatase showed no correlation with malignancy and was generally unremarkable except for high activity in osteoclasts, but was raised in two sarcomata that occurred after irradiation of giant-cell tumours. 5. A non-osteogenic fibroma and a fibrous cortical defect, though poorly represented in this series, are not uncommon; they sometimes lead to pathological fracture, but sarcoma is very rare in such lesions. They tend to show more alkaline phosphatase than fibrosarcoma but not the very high activity of fibrous dysplasia, which is related to its osteogenic potential. 6. Fibrous dysplasia most often presents in the five to fifteen age group but seldom leads to malignancy, though this may occur, usually as osteosarcoma, which has a similar high content of alkaline phosphatase. Fibrosarcoma is typically negative or very weak in this enzyme: the exceptional cases with high activity were tumours which were in part osteosarcoma. Generally the demonstration of high alkaline phosphatase activity in a fibroblastic lesion of bone, in the absence of trauma or inflammation, suggests the diagnosis of fibrous dysplasia

1. One hundred and seventy-nine cases of primary malignant bone tumour and giant-cell tumour seen at the Middlesex Hospital since 1925 are reviewed. Tumours arising from non-skeletal tissues in bone have been excluded. 2. The following histological classification is used. Osteosarcoma (osteoblast sarcoma): This tumour is not synonymous with osteogenic (bone-forming) sarcoma. The essential feature is the formation of osteoid tissue by malignant osteoblasts, with no intermediate matrix of cartilage or fibrous tissue. It is the most malignant bone tumour and only four of the thirty-two patients survived three years. Chondrosarcoma: These tumours are composed of cartilage, and some show secondary ossification. The behaviour of this group is related to the degree of cartilaginous differentiation. In general, compared with the osteosarcoma, it is of low-grade malignancy. More than half of the sixty-eight patients survived four years. Fibrosarcoma: The essential feature of this tumour is the production of collagen by malignant fibroblastic tumour cells. Tumours of this type invading the medullary cavity have an average prognosis between that of an osteosarcoma and a chondrosarcoma. Nine of the thirty-four patients survived three years. Spindle-cell sarcoma: These tumours are composed of spindle cells which produce no diagnostic matrix. In spite of the lack of differentiation the outlook is not hopeless. Six of the eleven patients survived for five years or more. Giant-cell tumour: This tumour is composed of a cellular stroma with diagnostic giant cells resembling osteoclasts. It is by no means a benign lesion, for half the tumours recurred after treatment and a quarter of the patients died with metastases. 3. The subdivision of primary malignant skeletal tumours into groups according to the histological pattern appears to be reflected in the behaviour of the individual tumours after treatment. The prognosis of each group has been stated in the appropriate sections

Objectives

The osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) balance is of the utmost importance in fracture healing. The aim of this study was therefore to investigate the impact of nonosteogenic factors on OPG and RANKL levels.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have emerged as potential predictive, prognostic, and therapeutic biomarkers, relevant to many pathophysiological conditions including limb immobilization, osteoarthritis, sarcopenia, and cachexia. Impaired musculoskeletal homeostasis leads to distinct muscle atrophies. Understanding miRNA involvement in the molecular mechanisms underpinning conditions such as muscle wasting may be critical to developing new strategies to improve patient management. MicroRNAs are powerful post-transcriptional regulators of gene expression in muscle and, importantly, are also detectable in the circulation. MicroRNAs are established modulators of muscle satellite stem cell activation, proliferation, and differentiation, however, there have been limited human studies that investigate miRNAs in muscle wasting. This narrative review summarizes the current knowledge as to the role of miRNAs in the skeletal muscle differentiation and atrophy, synthesizing the findings of published data.

Cite this article: Bone Joint Res 2020;9(11):798–807.

1. Alkaline and acid phosphatase, non-specific esterase and beta-glucuronidase have been estimated and demonstrated histochemically in a series of bone tumours and allied lesions, of which ten were osteogenic sarcomata, ten were giant-cell lesions, eleven were fibroblastic lesions and seven were tumours of cartilage. 2. Osteogenic sarcoma was found to be characterised by high levels of alkaline phosphatase, with rich staining for this enzyme in the tumour cells. Similar high levels of alkaline phosphatase were found in other bone-forming lesions, such as fibrous dysplasia, a giant-cell sarcoma with osteogenic matrix, and fracture callus. 3. Giant-cell lesions were characterised by high levels of acid phosphatase, and intense staining for this enzyme in the osteoclasts. These cells were also found to be rich in non-specific esterase (as shown by the alpha-naphthyl acetate method) and in beta-glucuronidase, but almost or entirely lacking in alkaline phosphatase. High levels of alkaline phosphatase were not found in giant-cell lesions except in relation to osteogenic matrix. 4. Fibroblastic tumours were characterised by moderate levels of all four enzymes, with little or no staining for phosphatases in the tumour cells; non-specific esterase was generally present in a proportion of the cells. 5. In certain lesions intermediate stages in the differentiation of fibroblasts to osteoblasts were found, notably in fibrous dysplasia, in which the biochemical change preceded the histological. In such lesions high total levels of alkaline phosphatase were found. 6. Cartilaginous tumours were characterised by low levels of all four enzymes, and little histochemical staining except in hypertrophied cells in areas of ossification. 7. It was found in general that the enzyme distributions in these neoplasms and other lesions reflected the findings in comparable reactive and growing normal tissues

Objectives

Many in vitro studies have investigated the mechanism by which mechanical signals are transduced into biological signals that regulate bone homeostasis via periodontal ligament fibroblasts during orthodontic treatment, but the results have not been systematically reviewed. This review aims to do this, considering the parameters of various in vitro mechanical loading approaches and their effects on osteogenic and osteoclastogenic properties of periodontal ligament fibroblasts.

Aims

Surgeons and most engineers believe that bone compaction improves implant primary stability without causing undue damage to the bone itself. In this study, we developed a murine distal femoral implant model and tested this dogma.

Objectives

The aim of this study was to provide a comprehensive understanding of alterations in messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) in cartilage affected by osteoarthritis (OA).

During the last decades, several research groups have used bisphosphonates for local application to counteract secondary bone resorption after bone grafting, to improve implant fixation or to control bone resorption caused by bone morphogenetic proteins (BMPs). We focused on zoledronate (a bisphosphonate) due to its greater antiresorptive potential over other bisphosphonates. Recently, it has become obvious that the carrier is of importance to modulate the concentration and elution profile of the zoledronic acid locally. Incorporating one fifth of the recommended systemic dose of zoledronate with different apatite matrices and types of bone defects has been shown to enhance bone regeneration significantly in vivo. We expect the local delivery of zoledronate to overcome the limitations and side effects associated with systemic usage; however, we need to know more about the bioavailability and the biological effects. The local use of BMP-2 and zoledronate as a combination has a proven additional effect on bone regeneration. This review focuses primarily on the local use of zoledronate alone, or in combination with bone anabolic factors, in various preclinical models mimicking different orthopaedic conditions.

Cite this article: I. Qayoom, D. B. Raina, A. Širka, Š. Tarasevičius, M. Tägil, A. Kumar, L. Lidgren. Anabolic and antiresorptive actions of locally delivered bisphosphonates for bone repair: A review. Bone Joint Res 2018;7:548–560. DOI: 10.1302/2046-3758.710.BJR-2018-0015.R2.

Bone is one of the most highly adaptive tissues in the body, possessing the capability to alter its morphology and function in response to stimuli in its surrounding environment. The ability of bone to sense and convert external mechanical stimuli into a biochemical response, which ultimately alters the phenotype and function of the cell, is described as mechanotransduction. This review aims to describe the fundamental physiology and biomechanisms that occur to induce osteogenic adaptation of a cell following application of a physical stimulus. Considerable developments have been made in recent years in our understanding of how cells orchestrate this complex interplay of processes, and have become the focus of research in osteogenesis. We will discuss current areas of preclinical and clinical research exploring the harnessing of mechanotransductive properties of cells and applying them therapeutically, both in the context of fracture healing and de novo bone formation in situations such as nonunion.

Cite this article: Bone Joint Res 2019;9(1):1–14.

1. Autografts, isografts and homografts of fibrocartilaginous callus were observed in the anterior chamber of the eye in rats. Proliferation of cartilage ceased, endochondral ossification followed, and the end-product was a new and complete ossicle with a cortex and a marrow cavity. The size and shape of the ossicle was determined by the size and shape of the sample of callus. Thus the callus in the eye performed the function of a cartilage model like that of the developing epiphysis or a healing fracture of a long bone. 2. Fibrocartilaginous callus, heavily labelled with . 3. H-thymidine, was transplanted to the eye twenty-four hours after the last injection, when there was little if any radioactive thymidine circulating in the blood. A few small chondrocytes with labelled nuclei persisted in the cores of new bone trabeculae, but the largest part of the labelled callus was resorbed and replaced by unlabelled new bone. 3. Homografts of labelled callus produced the same results as autografts at twenty-five days, but between twenty-five and forty-five days the donor cells were destroyed by the immune response of the host. 4. Isogenous transplants in host rats treated with . 3. H-thymidine between nine and thirteen days, when the callus was invaded by new blood vessels, produced many osteogenetic cells with labelled nuclei and made it possible to trace the origin of the new bone. The label appeared in the progenitor cells within twenty-four hours. While remaining thereafter in progenitor cells, it appeared also in osteoclasts (or chondroclasts) and osteoblasts in forty-eight to seventy-two hours, and in osteocytes in ninety-six to 120 hours. Chondrocytes did not proliferate and were not labelled in the eye. 5. Homogenous transplants in host rats treated with . 3. H-thymidine between five and one days before the operation also produced new bone, but contained no labelled osteoprogenitor or bone cells after twenty-five days in the eye. At forty-five days the donor tissue had been destroyed by the immune response of the host. 6. Devitalised callus was encapsulated in inflammatory connective tissue and scar. When the dead callus was absorbed by the capillaries of the host new bone formation by induction produced a scanty deposit as a delayed event in a few instances. 7. Irrespective of whether it originated in the donor or the host, a connective-tissue cell type that proliferated rapidly and became labelled with . 3. H-thymidine was identified as a progenitor cell. Differentiation and specialisation as osteoprogenitor cells occurred after the growth of blood vessels into the interior of the callus, and developed inside of excavation chambers in cartilage. Except that the interaction of the donor tissue and host cells leading to new bone formation by induction takes place in the interior of the excavation chamber, the biophysico-chemical mechanism is unknown

The experiments were performed to answer three main questions. These and our answers may be summarised as follows. What is the precise mechanism of healing of a raw bony surface in a joint? What cells are involved? Where do they originate?—In all the implant experiments and in the control series the fundamental mechanism of healing was similar. 1. A massive proliferation of fibroblasts occurred from the cut periosteum, from the cut joint capsule, and to a lesser extent from the medullary canal. 2. Fibroblasts grew centripetally in the first few weeks after operation, attempting to form a "fibroblast cap" to the cut bone end. 3. Fibroblasts of this cap near the cut bone spicules metamorphosed to become prechondroblasts, chondroblasts laying down cartilage matrix, and hypertrophied (alkaline phosphatase-secreting) chondrocytes lying in a calcified matrix. 4. This calcified cartilage matrix was invaded by dilated capillaries probably bearing osteoblasts which laid down perivascular (endochondral) bone. 5. Some of the cells of projecting bone spicules died and their matrix was eroded in the presence of many osteoclasts. 6. In the control experiments of simple excision of the radial head new bone was produced at the periphery only by processes (3) and (4). This sealed off the underlying peripheral cortical bone from the superficially placed peripheral articular surface of fibrocartilage. At about a year from operation the central portion of the articular surface was still formed of bare bone, or of bone spicules covered by a thin layer of irregularly arranged collagen fibres. The opposite capitular articular cartilage was badly eroded. Does the introduction of a dead cartilage implant over the raw bone end affect in any way the final constitution of the new articular surface?—In the implant experiments the new bone produced by processes (3) and (4) formed, after about a year, a complete cortical plate which entirely sealed off the cut end of the radius and left a superficially placed articular covering of smooth fibrocartilage, closely resembling a normal joint surface. The opposite capitular articular surface was normal. What is the final fate of such an implant?—Whale cartilage implants underwent replacement by fibroblasts and collagen fibres, and took about nine months to disappear. The cartilage of fixed autotransplants and homotransplants underwent similar gradual replacement, and took about the same time in each case. The dead bone, implanted in association with the cartilage in both cases, acted as a nidus for hyaline cartilage production by chondrocytes derived from fibroblasts. This cartilage underwent endochondral ossification. This observation suggests that induction by non-cellular osseous material is a factor in chondrification and ossification. All the implants functioned as temporary articular menisci or in some cases as temporary radial articular surfaces. They were always replaced by a permanent fibrocartilaginous meniscus, or a fibrocartilaginous articular surface. An implant did, in fact, always act as a temporary protecting cap and mould for the subjacent growth offibroblasts which was necessary for the production of a satisfactory new joint surface

Objectives

Bone fracture healing is regulated by a series of complex physicochemical and biochemical processes. One of these processes is bone mineralization, which is vital for normal bone development. Phosphatase, orphan 1 (PHOSPHO1), a skeletal tissue-specific phosphatase, has been shown to be involved in the mineralization of the extracellular matrix and to maintain the structural integrity of bone. In this study, we examined how PHOSPHO1 deficiency might affect the healing and quality of fracture callus in mice.

Objectives

Long bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done.

Aims

The aim of this study was to investigate the effects of preoperative bisphosphonate treatment on the intra- and postoperative outcomes of arthroplasty of the shoulder. The hypothesis was that previous bisphosphonate treatment would adversely affect both intra- and postoperative outcomes.