Tissue engineering advances seem to be more readily reported in other surgical specialties. The advances reported in orthopaedics in the past have really been limited to the rotator cuff or the anterior cruciate ligament. However, there are several advancements and opportunities in orthopaedic traumatology that involve tissue engineering. The main areas of interest include bone, scaffolds to make up for bone defects, cartilage, and soft tissue.
Emerging approaches to bone regeneration rely on principles of tissue engineering. This principle of guided tissue formation depends on access to osteogenic cells, relevant bone-forming growth factors or proteins, and a biomaterial or scaffold to provide an important osteoconductive environment to protect and host the process.
Developments in scaffold-based approaches provide new options for restoring form and function. These approaches will be discussed in a subsequent section. When considering the critical elements of a tissue engineering approach to bone regeneration (cells, growth factors, and scaffolding), clinical availability of cells is surprisingly the most advanced of all the elements; however, the in vivo activity of these cells still remains unclear.1
There are an overwhelming number of basic science studies demonstrating successful guided regeneration of bone using cell-based approaches, but the discrepancy between the basic science studies and clinical applications is vast. But, there is value in reviewing the basic research that will influence the critical translational studies and proof of concept work. Some of the most convincing work on large defect regeneration has been demonstrated in the rat critical femoral defect model.2–6 Although consistent regeneration can be achieved with recombinant growth factor augmentation,7 success with isolated cell-based approaches has been less consistent without the use of genetically modified cells or specific cell populations. Nauth et al have shown good results with endothelial progenitor cells.2 However, the feasibility of isolating and reintroducing a highly specific cell type (such as endothelial progenitor cells) in significant number at the point of care may not be practical, especially without external processing and modification.
Clinical successes with tissue engineering of bone with cell-based approaches are highly innovative, but also highly anecdotal.8 For example, craniomaxillofacial surgeons attempted guided bone regeneration of a mandible after tumor resection. This concept of an in vivo bioreactor approach created enthusiasm regarding the potential for customized and personalized bone regeneration. This concept involved implanting a custom scaffold filled with allograft and bone morphogenetic protein (BMP) into an ectopic muscular pouch, and then a later surgery to harvest this vascularized custom graft with an accompanying vascular pedicle. Although it successfully formed new bone, it functioned poorly and became infected. Limitations of this approach include the need for several surgical procedures, an extremely specialized skill set (microvascular anastomoses), and a patient with the resources to remain in a protracted and highly specialized treatment setting. For many trauma patients, this type of care is not practical or available. More than anything else, these initial attempts at bone regeneration have highlighted the need for simple and ideally single application solutions available at the point of care. In addition, any approach involving removal and modification of cells will meet challenging regulatory pathways.
Multiple types of approaches can provide cells currently. Concentrated bone marrow aspirate is widely available and used clinically in the treatment of delayed and nonunions. Previous work9 has defined critical threshold progenitor cell numbers for effective treatment, but the number of progenitors is not typically determined at the point of care which makes interpretation of this technique challenging.
Cells are also available in new viable allograft vehicles. These products typically contain allograft, demineralized bone matrix, and mesenchymal stem cells (MSCs). Testing of the products off the shelf has verified the presence of functionally competent MSC in comparable numbers to bone marrow aspirate.10 However, the presence of these cells in no way guarantees their viability and functionality. Although clinical studies do exist using these products in spine, foot, and craniomaxillofacial reconstructions, the results are difficult to interpret, as the studies are not prospective or comparative.
Lipoaspirates represent a newer, alternative cell source for tissue regeneration strategies. The technique (Lipogems; Lipogems International SpA, Milan, Italy) was developed to obtain microfragmented adipose tissue with an intact stromal vascular niche and MSCs with a high regenerative capacity. Like other technologies, cells can clearly be harvested with this technique. However, the bioactivity and capability of these cells to behave in vivo as osteogenic cells remains unverified. Dufrane et al11 attempted a proof of concept for bone regeneration using lipoaspirate-harvested cells, ex vivo expansion, DBM augmentation, and then preimplantation in bone regeneration cases. Their study only included a few patients and the results were ambiguous.
The final cell source under current investigation is cell isolation from the effluent/irrigant after use of the reamer irrigator aspirator (Depuy Synthes, West Chester, PA) system. There has been convincing demonstration of the presence of a large number of progenitor cells in the waste fluid irrigation from this reaming procedure.12 Current investigations are underway to explore the feasibility of harvesting these populations with increased efficiency from the high volume irrigant.
Although a number of cell sources are available to practicing physicians, significant questions remain. First, trauma applications will present host environments that are harsh to cells. The ability of these transplanted cells, regardless of source, to survive transplantation remains unclear. Second, the presence of the cells does not guarantee their function in terms of proliferation and expression of relevant growth factors for bone formation. Finally, despite extrapolation of studies from bone marrow concentrate aspirate, the optimal cell number for each application and cell type remains elusive. Nonetheless, a functional cell source will likely be a critical part of a successful tissue regeneration strategy and merits ongoing study.
Growth factors are also a critical part of approaches to tissue regeneration, and while clinical access to BMP has been in place for many years, little advance has occurred in improving clinical performance. Concerns about the safety of the current clinically available BMP product13 have demonstrated the need for new products with better safety profiles with fewer side effects. New growth factors are being evaluated. Many of these stimulate the canonical Wnt signally pathway. Nel-like molecule Nell-114 is an example of a potential future clinically relevant growth factor. It stimulates runx2 expression and is highly osteoinductive. Like any of these bioactive compounds, there will be significant challenges in the regulatory path to market. However, we will surely have access to highly inductive proteins for developing relevant tissue regeneration strategies.
In the end, clinically translatable approaches from the basic science realm are still lacking. Many basic science strategies for bone regeneration tend to work in small animal models but are challenging to be successful in larger models and humans. Efforts will continue, but it is clear that multiple factors are required to be clinically successful.
CAN WE GET A SCAFFOLD TO REALLY WORK?
Using autograft for large bone defects has its challenges such as limited available bone volume that fails to support the requirements of large defects, chronic pain at the graft site in nearly one-third of patients, and poor survival of transplanted cells motivate the need for alternative approaches to bone healing. Other strategies, including the delivery of potent recombinant osteoinductive proteins (eg, BMP-2),15 allograft bone, and natural and synthetic materials, also have their limitations. Presently, most scaffolds used in bone repair are used as graft extenders, capitalizing on the osteogenic potential of autologous biological materials (eg, bone graft, bone marrow aspirate, MSCs, reamer-irrigator-aspirator product) that are limited by donor variability in their potency. Thus, there is a critical need to enhance the stimulatory and reparative activity of scaffolds used to bridge bone defects and provide a more reproducible response in patients requiring intervention for bone repair.
When considering the development of scaffolds for bone repair, the long-held paradigm remains that scaffolds should exhibit similar mechanical properties to bone. A number of materials are clinically available to stabilize and bridge large bone defects including metals (eg, stainless steel) or bioceramics (eg, hydroxyapatite, tricalcium phosphate, and bioactive glasses). However, these nondegrading or slow-degrading materials possess mechanical properties that surpass native bone, which can result in bone resorption around the implant, formation of inflammatory wear debris, and additional revision procedures. As an alternative, scaffolds that are more compliant than host bone, formed of synthetic and natural polymers or other biological materials, may be useful. Furthermore, the use of such materials facilitates the incorporation of biological activity in the scaffold or transplant bone-forming cells that is more challenging to attain with other inert materials.
BMP-2, one of the most potent osteoinductive growth factors available, is an alternative to graft materials. The INFUSE Bone Graft (Medtronic) releases BMP-2 from an absorbable collagen sponge and induces osteogenic differentiation of surrounding responsive osteoprogenitor cells. Despite its efficacy in promoting bone formation, this product suffers from several contraindications including the risk of ectopic bone formation and increased levels of inflammation due to necessary high dosages.16 The bone-forming potential of BMP-2 is well-established, but the selection of carrier and achievable release kinetics have been implicated as key factors in its shortcomings. Although collagen is the predominant extracellular matrix (ECM) component of bone, this material does not permit sufficient control of protein retention, resulting in rapid local release from the sponge and the need for elevated, supraphysiological dosages. Numerous studies in the literature demonstrate outstanding efficacy to promote bone union of critical-sized defects when delivering low dosages of BMP-2 from alternative carriers such as hydrogels, macroporous scaffolds, and composites. These polymeric scaffolds enable improved control over growth factor release kinetics and diminish the shortcomings of current approaches.17,18 Such materials also permit the localized delivery of cells at the defect site or concomitant presentation of other bone-inducing factors.19,20 Thus, the use of BMP-2 may be expanded by selecting new scaffold materials.
Many scaffolds used in orthopaedic applications are inert by design. However, bone formation, which progresses from the edges toward the center of the defect, would benefit by strategies that enhance implant integration. Various strategies are under investigation including regulating surface roughness and pore diameter, controlling endogenous serum protein adhesion, and using biomineralized surfaces. Biomaterials used in tissue repair recapitulate the native biological niche, which is rich in ECM that regulates cell adhesion and differentiation. Our laboratory has designed endogenous, cell-secreted ECMs for use as a biomaterial and complexed these to other platforms for use in bone repair. The composition and function of the ECM can be controlled by the culture environment during manufacture,21 and the resulting biomaterial can be harvested, stored, and used as a coating on other materials.22,23 These ECMs can be formed from many cells of the mesenchymal lineage (ie, bone marrow, adipose tissue, and dermal fibroblasts).24 Importantly, the ECM facilitates improved local cell survival, enhanced osteogenic differentiation of host or exogenous bone-forming cells, and can sustain the phenotype of osteogenically induced cells on transplantation.23,25,26
Scaffold technologies are continuously progressing, regarding manufacturing techniques, mechanical properties, strategies to regulate mechanical properties and degradation, and delivery methods. However, because scaffolds are used to recapitulate the native bone tissue, even if for a short period, it is necessary to consider strategies to incorporate biological cues into these platforms to enhance their bioactivity, integration, and capacity to restore lost bone. The design and implantation of functional replacement bone tissue is a primary focus of the tissue engineering field. The transplantation of bone-forming cells on polymeric scaffolds has achieved somewhat limited success in preclinical and clinical use to date. However, the maturation of tissue-engineered implants by culture in bioreactors provides the opportunity to form a well-cellularized, robust bony implant. We and others have demonstrated the efficacy of this approach, demonstrating the culture durations of only 1 week are sufficient to achieve grafts with robust cell survival and bone formation in vivo.27,28 Thus, preconditioning of scaffolds in bioreactor systems is a promising pursuit for improving the bioactivity of bone implants.
In conclusion, scaffolds exhibit tremendous promise as alternatives to autologous bone graft. The composition, mechanical properties, and maturation by preconditioning before implantation provide exciting opportunities to regulate local presentation of potent osteoinductive cues, promote cell survival, and guide bone formation in situ.
Articular cartilage loss or significant comminution is devastating when managing articular fractures or injuries. Despite being able to accurately reconstruct the bony architecture, seeing an unreconstructable cartilage surface can crush your spirit. Most evidence for cartilage procedures come from Sports Medicine.29 Options are based on the size of the defect and joint involved. However, the FDA considers marrow stimulation procedures (MSPs)—that is, microfracture, the gold standard. Acute and delayed cartilage procedures exist. Acute procedures include debridement and lavage, marrow-stimulating procedures, osteochondral autografts, and modified marrow-stimulating procedures where allograft cartilage is combined with a marrow stimulating procedure. When considering all cartilage procedures in the knee (most commonly reported), 90 per 10,000 patients undergo surgery annually in the United States.30 Chondroplasty (debridement to a stable rim) is the performed twice as commonly as MSP and 50 times more common than autologous chondrocyte implantation and osteochondral autograft or allograft. Chondroplasty or MSP is the most common procedure performed for orthopaedic trauma by the authors as well.
It would be beneficial to have off-the-shelf cartilage augments as another option. Recently, modified MSPs have become more common because commercial cartilage allograft product availability has increased. These procedures account for less than 1% of the total procedures done annually. A modified MSP consists of doing microfracture and augmenting it with a cartilage allograft product such as DeNovo (ZimmerBiomet, Warsaw, IN) or BioCartilage (Arthrex, Naples, FL).
DeNovo is particulate juvenile cartilage allograft without an ECM that has been used in the shoulder, hip, knee, and ankle. Based on the manufacturer's recommendations, the subchondral bone should not be violated, and 1 pack of DeNovo should be used for every 2.5 cm2 of cartilage defect (www.zimmer.com). The cartilage graft position is maintained by applying fibrin glue. Farr et al31 published a case series using DeNovo for focal cartilage defects in the femoral condyles or trochlea and reported clinical outcomes, and magnetic resonance imaging (MRI) results in 25 patients with elective biopsy results in 8 of these patients. Clinical outcomes improved significantly over 2 years, MRI showed similar T2 signal to articular cartilage at 24 months, biopsy showed a mixture of hyaline and fibrocartilage with incorporation into the surrounding cartilage, and only one revision surgery with report of graft delamination.
BioCartilage is an ECM scaffold that is derived from allograft cartilage. It contains type 2 collagen, proteoglycans, and other cartilage growth factors. It is used in combination with microfracture to help direct autologous stem cells toward the cartilage line by providing an ECM with signal. It has been used in the elbow, hip, knee, and ankle. After being placed in the defect, the graft is secured with fibrin glue. It has a shelf life of 5 years. BioCartilage combined with platelet-rich plasma had significantly improved International Cartilage Repair Society repair score, repair-host histological scores, and improved MRI T2 findings when compared with microfracture alone in an equine full cartilage defect model at 13 months.32 Carter et al33 also showed improved MRI characteristics of the cartilage repair when BioCartilage was used instead of microfracture alone. This modified MSP has been helpful when intraoperative cartilage defects are identified (Fig. 1).
Scaffolds can also be used acutely to address chondral defects. Cartiform (Arthrex) is a commercially available product that can accommodate up to a 20 × 25-mm defect. It is derived from human hyaline cartilage and contains viable chondrocytes, ECM, and chondral proteins. It is cryopreserved and has a shelf life of 2 years. Only one case study is available that reports the technique for patellar chondral defects.34
Stem cells have been used in isolation and in combination with scaffolds in an effort to regenerate cartilage in mostly preclinical models. Goldberg et al35 did a systematic review of MSC use in cartilage repair and regeneration. They reviewed 252 studies including 100 in vitro, 111 animal, and 31 clinical studies. Cell source, number, and type varied significantly in the preclinical models, and 53 different scaffolds were used in 82% of the studies. There was a lack of correlation between the in vitro, animal, and clinical studies. Translational models need to be better to positively affect patients.
In orthopaedic trauma, larger defects (greater than 2.5 cm2) of the femoral head have been addressed acutely with frozen osteochondral allograft (OCA) and osteochondral autograft. Recently, the second case report of using frozen osteochondral femoral head allograft to address cartilage loss in a femoral head fracture was reported.36 Others have used osteochondral autograft plugs (mosaicplasty) harvested from either the ipsilateral knee or femoral head and transferred to the femoral head fracture chondral defects.37,38
However, on a delayed basis, cartilage can be restored with autogenous chondrocyte implantation (ACI) and matrix-induced autologous chondrocyte implantation (MACI), and with fresh osteochondral allografts. Both ACI and MACI require 2 surgeries—1 to harvest the chondrocytes and 1 for implantation. On the other hand, fresh osteochondral allografts require time to match a donor. Most of the clinical trials comparing techniques have come from the knee. A systematic review of knee cartilage techniques evaluated randomized controlled trials.39 ACI and MACI were found to be superior at 10 years when compared with microfracture alone and osteoarticular transfer system (OATS). ACI and MACI did better than microfracture for large defects (>4.5 cm2) as well.
Fresh osteochondral allografts (OCAs) are a viable option for larger defects if the patient is able to wait for a donor match. Indications for the technique vary by joint, but successful outcomes have been achieved in femoral head cartilage lesions greater than 2.5 cm2 with up to an 80% survival rate at a mean of 18 months.40,41 Mei et al42 report a graft survival rate of 86% at 2 years and 67% at 9 years. (Fig. 2) Using a larger graft, rather than several smaller grafts, has benefits. In a canine model, larger allografts seem to perform better functionally and histologically than smaller autografts or allografts.43 It is believed that there is less surface irregularity and less traumatic implantation with the larger grafts due to the larger surface area.
Acute cartilage defects or comminution remains a challenge in orthopaedic trauma. There does not seem to be 1 perfect solution for any joint. The perfect solution would be readily available, and contain live cartilage cells that could reliably heal to the host tissue, and predictably perform like hyaline cartilage. This option currently does not exist, but as described, there may be some allograft options that are available, despite not having significant clinical data in articular fractures. Furthermore, the use of stem cell augmentation and scaffolds seems to be primarily stuck in the preclinical phase. However, the paucity of options will help continue to drive surgeons and scientists to continue to strive for a solution.
Orthopaedic defects often present concomitantly with soft-tissue damage. Traumatic injury to surrounding muscle, tendon and ligament, skin, and nervous tissue can compromise the efficacy of therapeutic interventions and restoration of function. Autologous tissue grafts are the gold standard for treating soft-tissue damage, but there are substantial limitations associated with tissue availability for large defects. In some cases, transplanted tissues seldom maintain long-term viability and necessary function, as in muscle transplantation. Tissue engineering represents a promising approach to extend the amount of available autologous tissue or generate functional constructs that will integrate more efficiently with the host and address the shortfall of transplantable tissues.
Large-scale skin damage due to burns or trauma necessitates rapid intervention to prevent infection and reconstitute the skin's barrier function. Beyond skin grafting and simple wound dressings, there are a host of tissue-engineered skin products, which vary in complexity from simply biomaterial-based, engineered to avoid degradation, to autologous cellularized engineered skin. Numerous material-driven approaches are emerging to facilitate host cell ingrowth including electrospinning of degradable polymers.44 These materials mimic the morphology of native ECM and can be loaded with mitogenic and chemotactic growth factors to speed healing. Split-thickness grafts allow clinicians to extend the coverage of autologous grafts. As an alternative, bioreactors that stretch native skin over several days can stimulate cell and matrix production within the graft, resulting in a transplantable graft with significant increases in both area and volume without compromises in thickness as might be expected.45 Cell-based tissue engineering approaches can be used to deliver skin-forming cells to the site or encourage keratinocyte and endothelial cell migration into the wound. MSCs formed into cellular aggregates were entrapped in engineered fibrin gels that simultaneously upregulated prostaglandin E2 (PGE2) and vascular endothelial growth factor secretion to address dysregulated signaling of these pathways.46,47 Using a human skin equivalent, MSC spheroids accelerated wound closure in vitro compared to recombinant vascular endothelial growth factor through upregulation of the MSC secretome,48 providing an improved strategy for using autologous cells for skin tissue engineering.
Nerve deficits significantly affect function. Nerve transplantation from autologous or allogeneic sources provides 1 approach to restore lost function at the defect site after significant nerve damage. However, nerve activity must be sacrificed in another location or allogeneic nerves are harvested from cadaveric donors, representing a critical need for effective tissue engineered therapies. Numerous nerve conduits are under development to enable the delivery of biomolecular therapies or stem cells that can indirectly or directly participate in nerve bridging.49,50 Self-alignment of Schwann cells within a tethered type 1 collagen matrix, followed by removal of interstitial fluid by compression, produced a stable tissue-like biomaterial that recreated the aligned cellular and ECM architecture associated with nerve grafts. Evaluation of this device for repair of a critical-sized 15-mm gap revealed that engineered neural tissue supported robust neuronal regeneration across the gap at 8 weeks.51 However, much work remains to identify and deploy the appropriate cell sources and bioactive factors to speed nerve healing. This is particularly true in light of exciting preclinical data and the phase 1/2a clinical trial (NCT02302157, the SCiStar Study) using embryonic stem cell–derived oligodendrocyte progenitor cells to treat spinal cord injury.
Skeletal muscle can regenerate in response to minor injuries, but more severe injuries occurring due to disease or significant trauma can have a devastating effect on patient mobility and quality of life. Clinically, no therapeutic intervention exists that allows for full functional restoration. Autologous muscle transplants in the form of free muscle transfers can lead to donor site morbidity and inadequate perfusion in transplanted muscles, while injection of muscle cells at the damaged site has not provided significant benefit, likely due to poor cell survival or potentially rejection of transplanted cells.52 Furthermore, traditionally effective bone repair methods such as BMP-2 for preclinical segmental bone defects are ineffective in the presence of simultaneous volumetric muscle loss.53 Recent studies show that local delivery of MSCs from engineered hydrogels can promote muscle repair through endogenous trophic factor secretion that mobilizes host cells to regenerate and remodel damaged muscle.54 This approach can be enhanced by coupling the localized presentation of growth factors that induce local angiogenesis and sustain cell survival. The development of mechanically responsive biomaterials, which can be manipulated externally and noninvasively, can further stimulate muscle repair by promoting local compression that improves muscle regeneration and significantly increases contractile force.55 The use of such mechanically responsive substrates may have broad applications in soft-tissue engineering.
Traumatic defects of bone, cartilage, and soft tissue create significant challenges. If the bone does not heal, the articular surface remains irregular, or muscle, nerve, and skin are dysfunctional, there are significant functional problems. Although there a few clinically available regenerative methods for each, the basic science realm still holds the most promise. As we have discussed, the future is bright. However, it seems that the main issue is lack of translation from the small animal model to larger animal models, and humans.
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