Chella Hagmeijer

The in vitro feasibility of a new one-stage cell-based procedure for meniscus regeneration was demonstrated in chapter 4. An increase in extracellular matrix (ECM) production was shown using a combination of meniscus cells and mesenchymal stromal cells (MSCs) in a 20:80 ratio. It appeared the MSCs stimulated the meniscus cells to produce ECM using communication via gap junctions before disappearing from the coculture. These two cell types can be combined with a meniscus scaffold, the Collagen Meniscus Implant (CMI®), to provide an environment for tissue production. This study investigated different in vitro methods for seeding the cells into the meniscus scaffold, where static seeding onto a dry scaffold showed the highest cell density and the best cell distribution compared to seeding by injection into a wet scaffold. The results of these in vitro experiments laid the foundation for clinical applications of a one-stage cell-based meniscus regeneration procedure.

The in vitro results of the different seeding methods were assessed for their clinical applicability in a cadaveric study, described in chapter 5, where a one-stage arthroscopic procedure for meniscus regeneration was mimicked. First, rapid digestion of meniscus tissue with 0.0125% Liberase resulted in sufficient living meniscus cells for a 20:80 ratio with allogeneic MSCs during a one-stage procedure. Second, this study showed a high cell count inside of the scaffold implanted in vivo, a good cell distribution throughout the complete scaffold, and no effect on cell viability when the cells were statically seeded onto a dry CMI, which was arthroscopically implanted afterwards. First implanting the CMI and then injecting the cells into the wet scaffold showed significantly worse results for all three different outcome parameters.

Besides MSCs, growth factors show great potential for meniscus regeneration, especially platelet derived growth factor (PDGF), transforming growth factor β1 (TGF-β1), and a combination of growth factors found in platelet lysate (PL) as shown in chapter 6. These growth factors showed stimulation of migration, proliferation, and/or ECM production for both meniscus cells and MSCs. To implement the effect of growth factors in a clinical setting and improve the current treatment of meniscus regeneration using a CMI, we showed a method to functionalize the CMI for growth factor binding peptides. Cell ingrowth of both meniscus cells and MSCs into either the CMI functionalized for VEGF or the CMI coated with PRP resulted in a higher cell density inside of the CMI compared to the negative control, which indicates we can attract endogenous growth factors present in the knee joint without injecting recombinant exogenous growth factors.

In chapter 7 we developed and validated a new Patient Reported Outcome Measure (PROM), the Patient Approved Knee Assessment (PAKA), for patients undergoing sports related knee surgery. The PAKA was developed in collaboration with patients to better understand and report patient needs and outcomes after knee surgery, contributing to the movement towards patient-centered care. To measure the differences in outcome after newly developed treatment options, uniform outcome measures are needed. Besides objective radiographic outcome measures, subjective PROMs are widely used in orthopedic surgery, showing the increasing interest in patient involvement during treatment and rehabilitation. However, most of these PROMs are developed by medical physicians, who consider different symptoms and risk factors to be important compared to patients. Therefore, a patient centered PROM was developed in collaboration with patients to account for all their needs after surgical treatment.

General discussion

Good alignment of the knee can prevent osteoarthritis

The anterior cruciate ligament (ACL) prevents anterior translation of the tibia and plays an important role in providing rotational stability of the knee. After ACL injury, these stabilizing functions should be adopted by the menisci, femoral and tibial condylar geometry, and active muscle control. The medial meniscus especially serves as a secondary stabilizer of the knee because of its firm attachment to the tibia, preventing anterior tibial translation. However, in chronic knee injuries, tears of the medial meniscus are commonly seen, possibly leading to an increased instability of the knee joint, whereas lateral meniscus tears are often generated after an acute trauma of the knee.

Our study on the development of secondary meniscus tears after ACL injury showed a significantly lower rate of new meniscus tears in patients undergoing acute ACL reconstruction. This could indicate that by providing new stability to the knee using ACL reconstruction, there is a lower burden on the other stabilizers of the knee, preventing chronic damage. With ACL reconstruction preventing anterior tibial translation, and preserved meniscus function (load transmission, shock absorbance, and stability), there is no increased load on the articular cartilage which would decrease the chance of developing early osteoarthritis after traumatic injury of the knee.

This theory could especially be important in young and active patients since their rate of return to (rotating) sport is higher than that of the older population. However, our data demonstrated no differences in the frequency of secondary medial versus lateral meniscus tears for patients treated non-operatively for their ACL tears. On the other hand, the frequency of lateral meniscus tears was significantly higher in patients treated non-operatively compared to the patients treated with ACL reconstruction (ACLR), both acute and delayed. This high rate of lateral meniscus tears could be caused by a high percentage of new acute traumas in ACL deficient knees, even though it is less likely these older patients return to high demand sports. Thus, even in patients who are not performing contact and pivoting sports, an ACL reconstruction should be considered. This could prevent (new) meniscus tears and possible cartilage damage, which occur due to instability of the ACL-deficient knee. In addition, Sanders et al. 2017 showed a high incidence of symptomatic arthritis in patients with ACL tears treated non-operatively compared to matched peers without ACL injury, and a significantly higher risk for secondary meniscus tears, which supports our results and ideas on the importance of good alignment of the knee to prevent an unstable joint and thereby the development of early osteoarthrosis.

Non-operatively treated patients had a higher percentage of lateral meniscus injuries compared to the ACLR groups. This could mean new trauma had occurred due to instability of the knee, since lateral meniscus tears often present after acute trauma of the knee. Both ACLR groups had a higher percentage of medial meniscus tears, which could indicate that chronic inflammation and/or damage of the knee already began immediately after the acute trauma, although the stability of the knee was restored afterwards.

Taking the ACL injury out of the equation, multiple studies showed good objective and subjective clinical success after meniscus repair for both short-term and long-term follow-up in adult patients. Our study on isolated meniscus repair was the first study to describe long-term follow up in a pediatric and adolescent population. We described an overall failure rate of arthroscopic repair of isolated meniscus tears of 42% after 18 years of follow-up (14 of the 33 knees), where all the failures were reported before midterm follow-up (5.8 years). Complex tear types and a rim width of > 3 mm were found to be risk factors for the (early) failure of the meniscal repair. At long-term follow-up, no new failures were reported.

Meniscus repair attempts to restore the native shape of the meniscus, improving alignment of the knee and healing of the torn meniscus tissue. However, the exact biological mechanism of meniscus repair is unknown. Bansal et al 2017 showed data on meniscus repair at short term follow-up, but data on follow-up after one year in animal studies is very limited, and does not provide information on the primary healing process. Accordingly, information about the healing process after meniscus repair, type of formed tissue, and influence on the articular cartilage is not well known. In clinical trials, second-look arthroscopies after meniscus repair are uncommon as well, making it difficult to obtain evidence for complete healing of the meniscus tissue. One may hypothesize that just replacing the torn meniscus in its original position provides good alignment of the knee, even without complete biological healing.

Our results showed failure of meniscus repair only in the early stages. This could be the result of a re-tear caused by an acute re-injury of the biologically inferior fibrous scar tissue formed in the first phase after repair. Possible changes to rehabilitation programs could lead to better long-term outcomes. In addition, most failed repairs were complex tear types, indicating these tear types might not be suitable for repair. Coming up with good indications for meniscus repair can increase the success at long-term follow-up.

Based on the results of our studies, we recommend performing ACL reconstruction in the early stages after injury, and meniscus repair for simple and bucket-handle meniscus tears. These interventions can restore good alignment of the knee, prevent instability, and optimize loading of the articular cartilage. This could contribute to prevention of early-onset osteoarthritis after traumatic injury of the knee. For complex tear types, partial meniscectomy combined with a replacement of the meniscus using regenerative medicine might be a good option. To conclude, good alignment of the knee is necessary for all different treatment options. Without good alignment, repairing or regenerating the meniscus is not advantageous, because in these cases the instability of the knee joint is predictive for new damage to the meniscus and the development of osteoarthritis.

There is added value of regenerative medicine therapy in meniscus injury

The incidence of concomitant meniscus tears in ACL injuries is high; approximately 55-65% is reported in literature. In addition, it is well-known that the healing rate of meniscus repair is higher when this procedure is combined with an ACL reconstruction. The influence of bone marrow in the knee joint—due to the drilling of holes in ACL reconstruction—can play a role in the increased healing rate. Also, a higher level of growth factors, specifically platelet-derived growth factor (PDGF), was detected in the synovial fluid directly after ACL reconstruction compared to meniscectomy alone, because the drilling of the femoral and tibial tunnels may release bone marrow, creating an biologically advantageous environment. These two factors provide evidence for a place in regenerative medicine therapy in meniscus repair, using both MSCs and growth factors.

This thesis showed an increase in GAG and collagen production in cocultures of meniscus cells combined with MSCs, compared to monocultures of meniscus cells. Cocultures with a higher percentage of MSCs resulted in the highest production of ECM. Other studies for both meniscus tissue and articular cartilage showed similar results, indicating MSCs stimulate ECM production in combination with other cell types. Different pathways are described by which MSCs interact with other cell types and promote EMC production and proliferation. However, the exact mechanism of the role of MSCs in regenerative therapies is still unknown.

Although it was originally believed that MSCs would differentiate into the cell type they were cultured with, recent studies show that MSCs stimulate other cells to proliferate and produce new ECM, while they disappear. We have shown the same mechanism for MSCs in coculture with meniscal cells. By using male MSCs and real time PCR for a Y chromosomal gene, we showed that the male cells disappeared from the cocultures, while the total amount of cells remained approximately the same, suggesting the meniscus cells proliferated. Positive connexin-43 staining and exchange of cytosolic labelling suggest the formation of active gap junctions. Although not directly shown in this thesis, it is likely that other methods of communication were also used by the cells. It is known that MSCs can also communicate in an indirect way, paracrine signalling, by the secretion of trophic factors and extracellular vesicles. The direct contact by the cells likely just enhanced the communication potential by adding direct transfer of signalling factors in the cytosol by gap junctions.

In the literature, there is discussion about whether autologous or allogeneic MSCs should be used for regenerative therapy. The safety of allogeneic stem cells has been proven in several clinical trials. In addition, many studies assign the effect of MSCs primarily to their trophic cell properties; they produce extracellular vesicles, cytokines and growth factors that suppress the immune response by inhibiting B- and T-cell proliferation and monocyte maturation, and by promoting generation of regulatory T cells and M2 macrophages. Only one paper has made a direct comparison between autologous and allogeneic MSCs in favor of allogeneic MSCs. Other benefits are that allogeneic MSCs can be made as an ‘off-the-shelf’ product to use in a one-stage procedure, can thus also be administered in acute or emergency situations, are more cost-effective and less time-consuming. Therefore we want to use allogeneic MSCs for our future in vivo experiments. In patients with a meniscus tear, allogenic MSCs can potentially boost the regenerative effect in combination with autologous meniscus cells, producing a regenerated meniscus very similar to the native tissue, and restoring joint homeostasis by producing immunomodulatory signalling factors.

This thesis demonstrated that not only MSCs but also growth factors can play a part in regenerative medicine treatments. Both platelet-derived growth factor (PDGF), transforming growth factor beta 1 (TGF-β1), and platelet lysate (PL) increased proliferation and/or ECM production of meniscus cells and MSCs. The concept of functionalizing the CMI with growth factor binding peptide was demonstrated with vascular endothelial growth factor (VEGF). To optimize this procedure for meniscus regeneration, functionalization for other growth factor binding peptides is necessary. A gradient of different growth factor binding peptides throughout the meniscus would be ideal to stimulate the cells to different functions. Since PDGF promoted migration of both meniscus cells and MSCs, it would be convenient to functionalize the outer rim of the meniscus implant with growth factor binding peptides that would allow PDGF to attract cells from the native meniscus into the scaffold to promote new tissue formation. TGF-β1 also increases proliferation and ECM production of meniscus cells, which is of great use throughout the complete scaffold. Therefore, functionalization of the CMI with more than one growth factor to target multiple pathways can increase the regenerative capacity of the treatment.

Besides figuring out how to functionalize the meniscus implant with different growth factor binding peptides, the ideal concentration of growth factors is difficult to determine. There needs to be a balance between the right combination of growth factors (bound to the peptides) and paracrine factors secreted by MSCs to create a regenerative microenvironment. A wide range of dose-dependent concentrations of different growth factors is given in the literature. To use the growth factor binding peptides in clinics, we want to capture the endogenous growth factors of the knee joint and increase their effectiveness because of the higher local concentration at sites where that specific growth factor is needed. This biological effect can be partially regulated by adjusting the amount of growth factor binding peptides used for functionalization, but is also partially dependent on the amount of growth factors present in the knee joint. An animal study, using growth factor binding peptides for different growth factors conducive to meniscus regeneration, should be performed. Hevesi et al 2019 showed a model for ACLR in rabbits, using an ACL sleeve, that is useful to study functionalized biomaterial and determine the biological availablity of growth factors present in the knee joint.

Based on the studies and statements addressed in this discussion, we have proven regenerative medicine therapies can be of great added value for meniscus injuries. This is especially true in the more complex tear types, where the current treatment is not optimal. We can apply for a first-in-man clinical trial, using allogeneic MSCs in combination with autologous meniscus cells and a meniscus scaffold. Functionalization of a meniscus scaffold with growth factor binding peptides still needs to be further investigated in vitro before introducing it into clinical practice.

Patient-centered care is important

Nowadays patients are more and more up-to-date on symptoms of disease, treatments and experimental therapies. They want to be involved in their treatment process, leading to the importance of shared decision making. To guide patients through the different treatment options, and together decide what the best treatment is for that particular patient, the medical doctor needs to know what is important for patients in outcomes after surgery. Currently, the success of a treatment is based on clinical outcome measures reported by different PROMs, developed by medical doctors. This will not provide the best clinical outcome measures. A PROM developed in collaboration with patients can highlight the specific items that patients indicate as relevant for good rehabilitation and a successful treatment. The questionnaire developed in this thesis can provide this information in patients with sports related knee injuries. In addition, using one PROM for all patients within the same injury category allows for better comparison of clinical outcomes after surgery versus non-operative treatment. This will again contribute to better clinical data to inform patients and achieve transparent shared decision-making and patient-centered care.

Conclusions and future perspectives

This thesis demonstrated a strong role for meniscal repair after meniscus injury, provided that the indication is established correctly for patients. We showed that a stable knee joint with an intact ACL is less likely to (re)tear the meniscus. In addition, the pediatric and adolescent population showed an early and higher failure rate after meniscal repair in complex tear types with a rim width > 3mm. These findings indicate that meniscus repair shows better results in a stable knee joint (with an intact ACL). Besides, early failure of meniscus repair happens more often than late failure, which might be due to (too) early return to sport. Lastly, complex tear types might not be suitable for meniscus repair.

Solid guidelines can contribute to improved clinical outcomes after treatment for meniscus injury. Our recommendation for these guidelines include (1) combining ACL reconstruction with meniscus treatment; (2) using meniscus repair only in simple and bucket-handle tear types, with a rim width ≤2mm; (3) creating a conservative rehabilitation program after meniscus repair; (4) introducing a new intervention, meniscus regeneration, for complex tear types to prevent OA after partial meniscectomy.

This thesis reported good to excellent clinical outcomes after meniscus repair. However, meniscus repair is not suitable for all tear types. Based on the promising in vitro results of this thesis, a first-in-man clinical trial for meniscus regeneration should be performed. Regenerative medicine for meniscus tears can improve the clinical outcome for tear types in which repair is not an option. The Collagen Meniscus Implant (CMI) is FDA approved for clinical use, and allogeneic MSCs are proven to be safe for the use of cartilage regeneration.

The cell ratio of 20% allogenic MSCs and 80% autologous meniscus cells is proven to produce the highest amount of ECM, most comparable with native meniscus tissue. The amount of autologous meniscus needed for this ratio is clinically feasible to yield from harvested meniscus tissue, using Liberase. By statically seeding this cell combination onto the CMI, with fibrin glue as a carrier, good distribution and cell survival can be achieved, after which the scaffold can be arthroscopically implanted into the knee joint. A conservative rehabilitation program should be developed, patient progress should be monitored by using PROMs, MRI data should be obtained to monitor the degeneration of the scaffold and new tissue formation by augmented cells, and a second-look arthroscopy combined with a biopsy should be performed after one year. These outcome measures need to be combined to report the effectiveness of this new one-stage procedure for meniscus regeneration in young and active patients with an acute meniscus tear.

Personalized medicine is a growing field of orthopedic care and research. In regenerative medicine, porous materials are necessary to manufacture different type of scaffolds that provide mechanical stability and allow cell ingrowth to stimulate new tissue formation. It has already been shown that combining preoperative imaging with 3D printing of orthopedic implants (personalized medicine), results in improvement of surgical accuracy and clinical outcomes. In regenerative medicine, personally designed and 3D printed scaffolds based on preoperative imaging, can contribute to better clinical outcomes after surgery. This could lead to a shift in orthopedic surgery, from treating end-stage diseases such as osteoarthritis, to preventing end-stage diseases through treatments (i.e. meniscus and cartilage repair) using regenerative medicine.

How will this thesis change clinical practice?

1. We provided clear guidelines for when to use meniscal repair in the pediatric and adolescent population to prevent failure of treatment.
2. We demonstrated stability of the knee is of great importance. Therefore, in young and active patients with ACL injury (with or without concomitant meniscal tear), we recommend ACL reconstruction to prevent (secondary) meniscal tears and osteoarthritis.
3. We reported the in vitro and pre-clinical data for a new treatment in patients with a meniscal tear, meniscus regeneration. These data provide enough encouraging results to start a first-in-man clinical trial.
4. We developed and validated the Patient Approved Knee Assessment (PAKA) for patients with sports related knee injuries. This PROM can contribute to obtaining more relevant clinical outcome measures, better comparisons of different treatments, and moving towards patient-centered care.